Low-viscosity, high concentration evolocumab formulations and methods of making the same

ABSTRACT

Provided herein are formulations of PCSK9-binding polypeptides, such as those comprising evolocumab, that comprise N-acetyl arginine and have reduced viscosities when compared to formulations lacking N-acetyl arginine. Provided herein are also methods of formulating such compositions that are advantageous in that they conserve certain components. Such formulations comprising PCSK9-binding polypeptides can be administered to patients to treat and/or prevent PCSK9-related diseases, conditions, and disorders.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Pat. ApplicationNo. 62/462,266 filed Feb. 22, 2017, which is incorporated in itsentirety by reference herein.

SEQUENCE LISTING

The present application is being filed with a sequence listing inelectronic format. The sequence listing provided as a file titled,“A-2112-WO-PCT_sequence_listing_ST25.txt,” created Jan. 31, 2018, and is21 KB in size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

FIELD

The presented subject matter relates to the field of pharmaceuticalcompositions of evolocumab and other PCSK9-binding polypeptides andmethods of reducing viscosity of such compositions. Specifically, thepresented subject matter relates to pharmaceutical compositions ofevolocumab and other PCSK9-binding polypeptides comprising N-acetylarginine, and the use of N-acetyl arginine to decrease viscosity ofhigh-concentration evolocumab and other PCSK9-binding polypeptidesformulations. Furthermore, the disclosed subject matter presents methodsrelated to making such pharmaceutical compositions.

BACKGROUND

Therapeutic antibodies are formulated in solution for administration,such as parenteral injection. For products that are administeredsubcutaneously in self administration, formulations requiring deliveryvolumes greater than 1-2 milliliters are poorly tolerated. To solve thisissue, antibodies can be formulated at high concentrations (e.g., suchas 70 mg/mL to 210 mg/mL or greater), thus reducing the size of thedose.

Some highly concentrated antibody formulations can be challenging tomanufacture and administer, however. For example, in the formulation ofevolocumab (REPATHA®), a monoclonal antibody that binds PCSK9,concentrations of evolocumab above about 70 mg/mL have increasedviscosity. However, effective doses of evolocumab are 210 mg Q2W or 420mg Q4W. High viscosity formulations are not only difficult to handleduring manufacturing, including at the bulk and filling stages, but theyare also difficult to draw into a syringe and inject, makingadministration to the patient difficult and unpleasant.

To reduce viscosity of antibody formulations, unmodified arginine,glycine, serine, or proline amino acids have been added to antibodycompositions. For example, antibody formulations containing 80 mg/mL ofantibody and 75 mg/mL to about 125 mg/mL of arginine can be lyophilizedand reconstituted to 120-200 mg/mL; the final arginine concentrationscan be 431 mM to 718 mM (Morichika & Kameoka, 2007). While argininereduced the viscosity of the formulations when compared to controls(Morichika & Kameoka, 2007). Furthermore, the effect of arginine was notsufficient to reduce evolocumab viscosity to desired levels.

There is a need in the art to reduce the viscosity of evolocumab- andother PCSK9-binding polypeptides-containing formulations with compoundsthat are more efficient than arginine.

SUMMARY

In a first aspect, provided herein is a pharmaceutical compositioncomprising

-   a. a PCSK9-binding polypeptide that selected from the group    consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, l189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, l154, T1897, H193, E195, l196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, l369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR; and-   b. N-acetyl arginine,

wherein the pharmaceutical composition has a viscosity of at least lessthan about 80 cP. In such first aspect, the PCSK9-binding polypeptidecan be a monoclonal antibody that comprises a heavy chain polypeptidecomprising the following complementarity determining regions (CDRs):

-   a. heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs:7, 8, and 9, respectively; and-   b. light chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs:4, 5, and 6, respectively. Furthermore, in this first    aspect, the pharmaceutical composition can have a viscosity of at    least less than about 50 cP. The pharmaceutical composition can have    an osmolality of about 250 to about 400 mOsm/kg, such as about 300    mOsm/kg, or is isotonic to a human blood cell. The concentration of    the PCSK9-binding polypeptide can be from about 140 mg/mL to about    260 mg/mL, such as 210 mg/mL. The N-acetyl arginine can be present    at a concentration from about 25 mM to about 230 mM, such as 140 mM    to about 170 mM, or 140 mM. The pharmaceutical composition of this    aspect can further comprise a buffer, such as a buffer selected from    the group consisting of acetate, glutamate, histidine, and phosphate    buffers, or a combination thereof. The buffer can be present at a    concentration from about 5 mM to about 30 mM. In some cases, the    buffer is sodium acetate and is present at a concentration of about    10 mM. The pH of such pharmaceutical compositions can be from about    4.8 to about 6.9, such as a pH of about 5.4. The pharmaceutical    compositions of this aspect can further comprise a surfactant, such    as a surfactant selected from the group consisting of    polyoxyethylenesorbitan monooleate (polysorbate 80 or polysorbate    20), polyoxyethylene-polyoxypropylene block copolymer (Poloxamers    such as Pluronic® F-68 and other Pluronics®), Sorbitan alkyl esters    (Spans®) Polyethylene glycol octylphenyl ethers (Triton X-100),    Polyethylene glycol alkyl ethers (Brij), Polypropylene glycol alkyl    ethers, Glucoside alkyl ethers, and D-α-tocopherol polyethylene    glycol succinate (vitamin E TPGS). The surfactant can be present at    a concentration of about 0.0001% (w/v) to about 1% (w/v). In some    pharmaceutical compositions of this aspect, the surfactant is    polyoxyethylenesorbitan monooleate (polysorbate 80) and is present    at a concentration of about 0.01% (w/v). Furthermore, the    pharmaceutical compositions of this aspect can further comprise    proline, which can be present at a concentration of about 50 mM to    about 150 mM, such as 90 to 120 mM, or about 120 mM. In some cases,    the pharmaceutical composition of this first aspect can further    comprise an arginine salt, which can be present at a concentration    of about 25 mM to about 150 mM, such as about 50 mM to about 100 mM.    The arginine salt can be, for example, arginine-HCl, arginine    acetate, or arginine glutamate. In some cases, the arginine salt is    arginine HCl and is present at a concentration of about 50 mM. The    PCSK9-binding polypeptide can be stable for at least about two years    or even five years or more when stored at about -30° C. or colder in    the pharmaceutical compositions of this first aspect. At 5° C., the    PCSK9-binding polypeptide can be stable for at least about six    months to about 24 months or more in such pharmaceutical    compositions. At 25° C., the PCSK9-binding polypeptide can be stable    for at least about one month or longer, three months or longer, or    even six months or longer. At 40° C., the PCSK9-binding polypeptide    can be stable for at least one month or longer. The pharmaceutical    compositions of this first aspect can comprise high molecular weight    aggregates or oligomers of PCSK9-binding polypeptides at less than    about 3%, such as 2.5% or less, of the total PCSK9-binding    polypeptide concentration.

In a second aspect, disclosed herein is a pharmaceutical compositioncomprising

-   a. a PCSK9-binding polypeptide that selected from the group    consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, l189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, l154, T1897, H193, E195, l196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, l369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR; and    -   b. N-acetyl arginine;    -   c. an arginine salt;    -   d. a buffer; and    -   e. a surfactant    -   wherein the pharmaceutical composition has a viscosity of at        least less than about 80 cP.

In this second aspect, the PCSK9-binding polypeptide is a monoclonalantibody that comprises a heavy chain polypeptide comprising thefollowing complementarity determining regions (CDRs):

-   a. heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs:7, 8, and 9, respectively; and-   b. light chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs:4, 5, and 6, respectively.

The pharmaceutical compositions of this second aspect can have aviscosity of at least less than about 50 cP. The pharmaceuticalcomposition can have an osmolality of about 250 to about 400 mOsm/kg,such as about 300 mOsm/kg, or is isotonic to a human blood cell. Theconcentration of the PCSK9-binding polypeptide can be from about 140mg/mL to about 260 mg/mL, such as 210 mg/mL. The N-acetyl arginine canbe present at a concentration from about 25 mM to about 230 mM, such as140 mM to about 170 mM, or 140 mM. The pharmaceutical composition ofthis aspect can further comprise a buffer, such as a buffer selectedfrom the group consisting of acetate, glutamate, histidine, andphosphate buffers, or a combination thereof. The buffer can be presentat a concentration from about 5 mM to about 30 mM. In some cases, thebuffer is sodium acetate and is present at a concentration of about 10mM. The pH of such pharmaceutical compositions can be from about 4.8 toabout 6.9, such as a pH of about 5.4. The pharmaceutical compositions ofthis aspect can further comprise a surfactant, such as a surfactantselected from the group consisting of polyoxyethylenesorbitan monooleate(polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropyleneblock copolymer (Poloxamers such as Pluronic® F-68 and otherPluronics®), Sorbitan alkyl esters (Spans®) Polyethylene glycoloctylphenyl ethers (Triton X-100), Polyethylene glycol alkyl ethers(Brij), Polypropylene glycol alkyl ethers, Glucoside alkyl ethers, andD-α-tocopherol polyethylene glycol succinate (vitamin E TPGS). Thesurfactant can be present at a concentration of about 0.0001% (w/v) toabout 1% (w/v). In some pharmaceutical compositions of this aspect, thesurfactant is polyoxyethylenesorbitan monooleate (polysorbate 80) and ispresent at a concentration of about 0.01% (w/v). Furthermore, thepharmaceutical compositions of this aspect can further comprise proline;the proline can be present at a concentration of about 50 mM to about150 mM, such as 90 to 120 mM, or about 120 mM. In some cases, thepharmaceutical composition of this second aspect can further comprise anarginine salt, which can be present at a concentration of about 25 mM toabout 150 mM, such as about 50 mM to about 100 mM. The arginine salt canbe, for example, arginine-HCl, arginine acetate, or arginine glutamate.In some cases, the arginine salt is arginine HCl and is present at aconcentration of about 50 mM. The PCSK9-binding polypeptide can bestable for at least about two years or even five years or more whenstored at about -30° C. or colder in the pharmaceutical compositions ofthis second aspect. At 5° C., the PCSK9-binding polypeptide can bestable for at least about six months to about 24 months or more in suchpharmaceutical compositions. At 25° C., the PCSK9-binding polypeptidecan be stable for at least about one month or longer, three months orlonger, or even six months or longer. At 40° C., the PCSK9-bindingpolypeptide can be stable for at least one month or longer. Thepharmaceutical compositions of this second aspect can comprise highmolecular weight aggregates or oligomers of PCSK9-binding polypeptidesat less than about 3%, such as 2.5% or less, of the total PCSK9-bindingpolypeptide concentration.

In a third aspect, disclosed herein is a pharmaceutical compositioncomprising

-   a. a PCSK9-binding polypeptide that at a concentration of about    195-225 mg/mL selected from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, l189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, l154, T1897, H193, E195, l196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, l369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR;-   b. N-acetyl arginine present at a concentration of about 140 mM;-   c. arginine HCl present at a concentration of about 50 mM;-   d. polyoxyethylenesorbitan monooleate (polysorbate 80) at a    concentration of from about 0.005% (w/v) to about 0.015% (w/v); and-   e. sodium acetate at a concentration of about 10 mM.

In this third aspect, the pharmaceutical composition can have a pH ofabout 5.1 to about 5.7, such as a pH of about 5.4. The pharmaceuticalcomposition can have a viscosity of at least less than about 50 cP.

In a fourth aspect, disclosed herein is a pharmaceutical compositioncomprising

-   a. a PCSK9-binding polypeptide that at a concentration of about    195-225 mg/mL selected from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, l189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, l154, T1897, H193, E195, l196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, l369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and-   b. wherein the epitope of the monoclonal antibody further overlaps    with a site to which an EGF-A domain of LDLR;-   c. N-acetyl arginine present at a concentration of about 140 mM;-   d. arginine HCl present at a concentration of about 63 mM;-   e. polyoxyethylenesorbitan monooleate (polysorbate 80) at a    concentration of about 0.005% (w/v) to about 0.015%; and-   f. sodium acetate at a concentration of about 10 mM.

In this fourth aspect, the pharmaceutical composition can have a pH ofabout 5.1 to about 5.7, such as a pH of about 5.4. The pharmaceuticalcomposition can have a viscosity of at least less than about 80 cP.

In a fifth aspect, disclosed herein is a pharmaceutical compositioncomprising

-   a. a PCSK9-binding polypeptide that at a concentration of about    195-225 mg/mL selected from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, l189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, l154, T1897, H193, E195, l196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, l369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR;-   b. N-acetyl arginine present at a concentration of about 155 mM;-   c. arginine HCl present at a concentration of about 70 mM;-   d. polyoxyethylenesorbitan monooleate (polysorbate 80) at a    concentration of about 0.005% (w/v) to about 0.015% (w/v); and-   e. sodium acetate at a concentration of about 10 mM. In this fifth    aspect, the pharmaceutical composition can have a pH of about 5.1 to    about 5.7, such as a pH of about 5.4. The pharmaceutical composition    can have a viscosity of at least less than about 45 cP.

In a sixth aspect, disclosed herein is a pharmaceutical compositioncomprising

-   a. a PCSK9-binding polypeptide that at a concentration of about    195-225 mg/mL selected from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, l189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, l154, T1897, H193, E195, l196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, l369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR;-   b. N-acetyl arginine present at a concentration of about 170 mM;-   c. arginine HCl present at a concentration of about 63 mM;-   d. polyoxyethylenesorbitan monooleate (polysorbate 80) at a    concentration of about 0.005% (w/v) to about 0.015%; and-   e. sodium acetate at a concentration of about 10 mM.

In this sixth aspect, the pharmaceutical composition can have a pH ofabout 5.1 to about 5.7, such as a pH of about 5.4. The pharmaceuticalcomposition can have a viscosity of at least less than about 60 cP.

In a seventh aspect, disclosed herein is a pharmaceutical compositioncomprising

-   a. a PCSK9-binding polypeptide that at a concentration of about    195-225 mg/mL selected from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, l189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, l154, T1897, H193, E195, l196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, l369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR;-   b. N-acetyl arginine present at a concentration of about 155 mM;-   c. proline present at a concentration of about 120 mM;-   d. polyoxyethylenesorbitan monooleate (polysorbate 80) at a    concentration of about 0.005% (w/v) to about 0.015% (w/v); and-   e. sodium acetate at a concentration of about 10 mM. In this seventh    aspect, the pharmaceutical composition can have a pH of about 5.1 to    about 5.7, such as a pH of about 5.4. The pharmaceutical composition    can have a viscosity of at least less than about 60 cP.

In these third through seventh aspects, the PCSK9-binding polypeptidecan be stable for at least about two years or even five years or morewhen stored at about -30° C. or colder in the pharmaceuticalcompositions of these aspects. At 5° C., the PCSK9-binding polypeptidecan be stable for at least about six months to about 24 months or morein such pharmaceutical compositions. At 25° C., the PCSK9-bindingpolypeptide can be stable for at least about one month or longer, threemonths or longer, or even six months or longer. At 40° C., thePCSK9-binding polypeptide can be stable for at least one month orlonger. The pharmaceutical compositions of these aspects can comprisehigh molecular weight aggregates or oligomers of PCSK9-bindingpolypeptides at less than about 3%, such as 2.5% or less, of the totalPCSK9-binding polypeptide concentration.

In any of the preceding aspects, the pharmaceutical composition can be aliquid.

In an eighth aspect, disclosed herein is a method of treating a subjectin need thereof, comprising administering the pharmaceutical compositionof any of the preceding seven aspects.

In a ninth aspect, disclosed herein is a kit comprising a pharmaceuticalcomposition of any of the first through seventh aspects and a deliverydevice. The delivery device can be selected from the group consisting ofa syringe, an injector pen, a body injector, and an autoinjector. Thekit can further comprise instructions for administering thepharmaceutical composition using the delivery device.

In a tenth aspect, disclosed herein is a method of preparing aPCSK9-binding polypeptide pharmaceutical composition comprising at least140 mg/mL of PCSK9-binding polypeptide, comprising adding to apharmaceutical composition comprising the PCSK9-binding polypeptide aneffective amount of N-acetyl arginine, such that the viscosity of thepharmaceutical composition is reduced when compared to thepharmaceutical composition lacking the N-acetyl arginine, and whereinthe PCSK9-binding polypeptide is selected from the group consisting of:

-   a. a monoclonal antibody comprising a heavy chain polypeptide having    an amino acid sequence of SEQ ID NO:1 and a light chain polypeptide    having an amino acid sequence of SEQ ID NO:2 (evolocumab), or an    antigen-binding fragment thereof;-   b. a monoclonal antibody that competes with evolocumab for binding    to PCSK9;-   c. a monoclonal antibody, comprising:    -   i. a heavy chain polypeptide comprising the following        complementarity determining regions (CDRs): a heavy chain CDR1        that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain CDR2 that        is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain CDR3 that is a        CDR3 in SEQ ID NOs:14 or 16, and    -   ii. a light chain polypeptide comprising the following CDRs: a        light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a light        chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a light chain        CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;-   d. a monoclonal antibody that binds to at least one of the following    residues of PCSK9, the PCSK9 comprising an amino acid sequence of    SEQ ID NO:3: S153, D188, l189, Q190, S191, D192, R194, E197, G198,    R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377,    F379, l154, T1897, H193, E195, l196, M201, V202, C223, T228, S235,    G236, A239, G244, M247, l369, S372, C375, C378, R237, and D238; and-   e. a monoclonal antibody that binds to PCSK9 at an epitope on PCSK9    that overlaps with an epitope that is bound by an antibody that    comprises:    -   i. a heavy chain variable region of the amino acid sequence in        SEQ ID NO:1; and    -   ii. a light chain variable region of the amino acid sequence in        SEQ ID NO:2, and    -   iii. wherein the epitope of the monoclonal antibody further        overlaps with a site to which an EGF-A domain of LDLR.

In this tenth aspect, the viscosity of the pharmaceutical composition isless than about 80 cP, or less than about 50 cP. The pharmaceuticalcomposition of method of this aspect can have an osmolality of about 250to about 400 mOsm/kg, such as about 300 mOsm/kg, or is isotonic to ahuman blood cell. The concentration of the PCSK9-binding polypeptide canbe from about 140 mg/mL to about 260 mg/mL, such as 210 mg/mL. TheN-acetyl arginine can be present at a concentration from about 25 mM toabout 230 mM, such as 140 mM to about 170 mM, or 140 mM. Thepharmaceutical composition of this aspect can further comprise a buffer,such as a buffer selected from the group consisting of acetate,glutamate, histidine, and phosphate buffers, or a combination thereof.The buffer can be present at a concentration from about 5 mM to about 30mM. In some cases, the buffer is sodium acetate and is present at aconcentration of about 10 mM. The pH of such pharmaceutical compositionscan be from about 4.8 to about 6.9, such as a pH of about 5.4. Thepharmaceutical compositions of this aspect can further comprise asurfactant, such as a surfactant selected from the group consisting ofpolyoxyethylenesorbitan monooleate (polysorbate 80 or polysorbate 20),polyoxyethylene-polyoxypropylene block copolymer (Poloxamers such asPluronic® F-68 and other Pluronics®), Sorbitan alkyl esters (Spans®)Polyethylene glycol octylphenyl ethers (Triton X-100), Polyethyleneglycol alkyl ethers (Brij), Polypropylene glycol alkyl ethers, Glucosidealkyl ethers, and D-α-tocopherol polyethylene glycol succinate (vitaminE TPGS). The surfactant can be present at a concentration of about0.0001% (w/v) to about 1% (w/v). In some pharmaceutical compositions ofthis aspect, the surfactant is polyoxyethylenesorbitan monooleate(polysorbate 80) and is present at a concentration of about 0.01% (w/v).Furthermore, the pharmaceutical compositions of this aspect can furthercomprise proline; the proline can be present at a concentration of about50 mM to about 150 mM, such as 90 to 120 mM, or about 120 mM. In somecases, the pharmaceutical composition of this tenth aspect can furthercomprise an arginine salt, which can be present at a concentration ofabout 25 mM to about 150 mM, such as about 50 mM to about 100 mM. Thearginine salt can be, for example, arginine-HCl, arginine acetate, orarginine glutamate. In some cases, the arginine salt is arginine HCl andis present at a concentration of about 50 mM. The PCSK9-bindingpolypeptide can be stable for at least about two years or even fiveyears or more when stored at about -30° C. or colder in thepharmaceutical compositions of this tenth aspect. At 5° C., thePCSK9-binding polypeptide can be stable for at least about six months toabout 24 months or more in such pharmaceutical compositions. At 25° C.,the PCSK9-binding polypeptide can be stable for at least about one monthor longer, three months or longer, or even six months or longer. At 40°C., the PCSK9-binding polypeptide can be stable for at least one monthor longer. The pharmaceutical compositions of this tenth aspect cancomprise high molecular weight aggregates or oligomers of PCSK9-bindingpolypeptides at less than about 3%, such as 2.5% or less, of the totalPCSK9-binding polypeptide concentration.

In an eleventh aspect, disclosed herein is a method of formulating atherapeutic polypeptide, comprising

-   a. a first concentration step, wherein the polypeptide in a first    solution is concentrated;-   b. a first solution exchange step, wherein the concentrated    polypeptide in the first solution is exchanged into a second    solution comprising N-acetyl arginine using diafiltration;-   c. a second concentration step, wherein the polypeptide in the    second solution is concentrated;-   d. a second solution exchange step, wherein the polypeptide in the    concentrated second solution is exchanged into a third solution    comprising N-acetyl arginine using diafiltration; and-   e. a third concentration step, wherein the polypeptide in the third    solution is concentrated;

wherein the therapeutic polypeptide comprises a PCSK9-bindingpolypeptide that blocks binding of PCSK9 to LDLR and is selected fromthe group consisting of:

-   i. a monoclonal antibody comprising a heavy chain having an amino    acid sequence of SEQ ID NO:1 and a light chain having an amino acid    sequence of SEQ ID NO:2 (evolocumab), or an antigen-binding fragment    thereof;-   ii. a monoclonal antibody that competes with evolocumab for binding    to PCSK9;-   iii. a monoclonal antibody, comprising:    -   1. a heavy chain polypeptide comprising the following        complementarity determining regions (CDRs): a heavy chain CDR1        that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain CDR2 that        is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain CDR3 that is a        CDR3 in SEQ ID NOs:14 or 16, and    -   2. a light chain polypeptide comprising the following CDRs: a        light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a light        chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a light chain        CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;-   iv. a monoclonal antibody that binds to at least one of the    following residues of PCSK9, the PCSK9 comprising an amino acid    sequence of SEQ ID NO:3: S153, D188, l189, Q190, S191, D192, R194,    E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376,    T377, F379, l154, T1897, H193, E195, l196, M201, V202, C223, T228,    S235, G236, A239, G244, M247, l369, S372, C375, C378, R237, D238;-   v. a monoclonal antibody that binds to PCSK9 at an epitope on PCSK9    that overlaps with an epitope that is bound by an antibody that    comprises:    -   1. a heavy chain variable region of the amino acid sequence in        SEQ ID NO:1; and    -   2. a light chain variable region of the amino acid sequence in        SEQ ID NO:2, and    -   3. wherein the epitope of the monoclonal antibody further        overlaps with a site to which an EGFa domain of LDLR.

In the methods of this eleventh aspect, the PCSK9-binding polypeptidethat blocks binding of PCSK9 to LDLR can be a monoclonal antibody thatcomprises a heavy chain polypeptide comprising the followingcomplementarity determining regions (CDRs):

-   a. heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs: 7, 8, and 9, respectively; and-   b. light chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs: 4, 5, and 6, respectively.

Furthermore, in this eleventh aspect, before the third concentrationstep, the temperature of the solution comprising the polypeptide can beincreased from about 25° C. to about 37° C. Also, the first solutionexchange step can be accomplished using at least three diavolumes of thesecond solution. In some sub-aspects of this eleventh aspect, the secondsolution exchange step is accomplished using at least four diavolumes ofthe third solution. In other sub-aspects, the initial concentration ofthe therapeutic protein is about 11 mg/mL or less. Additionally, thetherapeutic polypeptide concentration can be increased from about 3- toabout 7-fold, such as, for example, where the increased concentration ofthe polypeptide is from about 35 mg/mL to about 70 mg/mL. In somesub-aspects, in the second concentration step, the therapeuticpolypeptide concentration is increased from about 2- to 4-fold from thefirst concentration step, such as to about 140 mg/mL, for example. Inthe third concentration step, the therapeutic polypeptide concentrationcan be increased from about 1.5- to about 2-fold from the secondconcentration step, such as to about 260 mg/mL. The therapeuticpolypeptide can therefore have a final concentration that is at leastabout 19-20-fold more concentrated than the initial concentration of thetherapeutic polypeptide, such as about 210 mg/mL. The concentrationsteps can comprise fed-batch ultrafiltration; furthermore, the secondsolution and the third solution can be identical. For example, thesecond or third solution comprising N-acetyl arginine can comprise anarginine salt and a buffer, wherein, for example, the N-acetyl arginineis present at a concentration of about 25 mM to about 230 mM; thearginine salt is Arg HCl, Arg acetate, or Arg glutamate and is presentat a concentration of about 25 mM to about 150 mM; and the buffer is asodium acetate buffer at a concentration of about 5 mM to about 30 mM.In other sub-aspects, the N-acetyl arginine is present at aconcentration of about 140 to about 170 mM; the Arg HCl, Arg acetate, orArg glutamate is present at a concentration of about 63 to about 70 mMand the sodium acetate buffer is present at a concentration of about 10mM. In yet other sub-aspects, the N-acetyl arginine is present at aconcentration of about 140 mM, the Arg HCl, Arg acetate, or Argglutamate is present at a concentration of about 63 mM, the sodiumacetate buffer is present at a concentration of about 10 mM. In furthersub-aspects, the N-acetyl arginine is present at a concentration ofabout 155 mM, the Arg HCl, Arg acetate, or Arg glutamate is present at aconcentration of about 70 mM, the sodium acetate buffer is present at aconcentration of about 10 mM. In yet other sub-aspects, the N-acetylarginine is present at a concentration of about 170 mM, the Arg HCl, Argacetate, or Arg glutamate is present at a concentration of about 63 mM,the sodium acetate buffer is present at a concentration of about 10 mM.Furthermore, the compositions can further comprise proline, wherein theproline is present at a concentration of about 50 mM to about 150 mM.The second or third solution can have a pH from about 4.8 to about 6.9,such as 5.4. In the first and second solution exchange steps, adiafiltration membrane can be used having at least one characteristicselected from the group consisting of:

-   a. mesh openings that are greater than about 350 µm but less than or    equal to about 500 µm;-   b. an open area that is greater than about 32% but less than or    equal to about 36% of the membrane area;-   c. a mesh count of less than about 16.2 n/cm but greater than or    equal to about 12.2 n/cm;-   d. a wire diameter that is greater than about 270 µm but less than    or equal to about 340 µm;-   e. a basis weight that is greater than about 160 g/m² but less than    or equal to 180 g/m²;-   f. a thickness greater than about 515 µm but less than or equal to    about 610 µm;-   g. a membrane load of greater than about 1138.1 g/m² but less than    or equal to about 1919.3 g/m²; and-   h. a maximum feed pressure of about 60 psi.

Furthermore, surfactant can added to the third solution after beingconcentrated, such as a surfactant selected from the group consisting ofpolyoxyethylenesorbitan monooleate (polysorbate 80 or polysorbate 20),polyoxyethylene-polyoxypropylene block copolymer (Poloxamers such asPluronic® F-68 and other Pluronics®), Sorbitan alkyl esters (Spans®)Polyethylene glycol octylphenyl ethers (Triton X-100), Polyethyleneglycol alkyl ethers (Brij), Polypropylene glycol alkyl ethers, Glucosidealkyl ethers, and D-α-tocopherol polyethylene glycol succinate (vitaminE TPGS). The surfactant can be present at a concentration of about0.0001% (w/v) to about 1% (w/v). In some pharmaceutical compositions ofthis aspect, the surfactant is polyoxyethylenesorbitan monooleate(polysorbate 80) and is present at a concentration of about 0.01% (w/v).

In a twelfth aspect, disclosed herein is a method of formulating atherapeutic polypeptide, comprising

-   a. a first concentration step, wherein the polypeptide in a first    solution is concentrated using fed-batch ultrafiltration;-   b. a first solution exchange step, wherein the concentrated    polypeptide in the first solution is exchanged into a second    solution comprising N-acetyl arginine, arginine salt, and a buffer,    using diafiltration and three diavolumes of the second solution;-   c. a second concentration step, wherein the polypeptide in the    second solution is concentrated using fed-batch ultrafiltration;-   d. a second solution exchange step, wherein the polypeptide in the    concentrated second solution is exchanged into third solution    comprising N-acetyl arginine, arginine salt, and a buffer using    diafiltration and four diavolumes of the third solution;-   e. the temperature of the solution comprising the polypeptide is    increased from about 25° C. to about 37° C. after the second    solution exchange step; and-   f. a third concentration step, wherein the polypeptide is further    concentrated using fed-batch ultrafiltration concentration;

wherein in the first and second solution exchange steps, a diafiltrationmembrane is used having at least one characteristic selected from thegroup consisting of:

-   a. mesh openings that are greater than about 350 µm but less than or    equal to about 500 µm;-   b. an open area that is greater than about 32% but less than or    equal to about 36% of the membrane area;-   c. a mesh count of less than about 16.2 n/cm but greater than or    equal to about 12.2 n/cm;-   d. a wire diameter that is greater than about 270 µm but less than    or equal to about 340 µm;-   e. a basis weight that is greater than about 160 g/m² but less than    or equal to 180 g/m²;-   f. a thickness greater than about 515 µm but less than or equal to    about 610 µm;-   g. a membrane load of greater than about 1138.1 g/m² but less than    or equal to about 1919.3 g/m²; and-   h. a maximum feed pressure of about 60 psi; and-   wherein the therapeutic polypeptide comprises a PCSK9-binding    polypeptide that blocks binding of PCSK9 to LDLR and is selected    from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain having an        amino acid sequence of SEQ ID NO:1 and a light chain having an        amino acid sequence of SEQ ID NO:2 (evolocumab), or an        antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, l189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, D238, K243, S373,        D374, S376, T377, F379, l154, T1897, H193, E195, l196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, l369, S372,        C375, C378, R237, D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGFa domain of LDLR.

Furthermore, in this twelfth aspect, the first solution exchange stepcan be accomplished using at least three diavolumes of the secondsolution. In some sub-aspects of this twelfth aspect, the secondsolution exchange step is accomplished using at least four diavolumes ofthe third solution. In other sub-aspects, the initial concentration ofthe therapeutic protein is about 11 mg/mL or less. Additionally, thetherapeutic polypeptide concentration can be increased from about 3- toabout 7-fold, such as, for example, where the increased concentration ofthe polypeptide is from about 35 mg/mL to about 70 mg/mL. In somesub-aspects, in the second concentration step, the therapeuticpolypeptide concentration is increased from about 2- to 4-fold from thefirst concentration step, such as to about 140 mg/mL, for example. Inthe third concentration step, the therapeutic polypeptide concentrationcan be increased from about 1.5- to about 2-fold from the secondconcentration step, such as to about 260 mg/mL. The therapeuticpolypeptide can therefore have a final concentration that is at leastabout 19-20-fold more concentrated than the initial concentration of thetherapeutic polypeptide, such as about 210 mg/mL. The concentrationsteps can comprise fed-batch ultrafiltration; furthermore, the secondsolution and the third solution can be identical. The N-acetyl argininepresent at a concentration of about 25 mM to about 230 mM; the argininesalt can be Arg HCl, Arg acetate, or Arg glutamate, wherein the Arg HCl,Arg acetate, or Arg glutamate is present at a concentration of about 25mM to about 150 mM; and the buffer is a sodium acetate buffer at aconcentration of about 5 mM to about 30 mM. In other sub-aspects, theN-acetyl arginine is present at a concentration of about 140 to about170 mM; the Arg HCl, Arg acetate, or Arg glutamate is present at aconcentration of about 63 to about 70 mM and the sodium acetate bufferis present at a concentration of about 10 mM. In yet other sub-aspects,the N-acetyl arginine is present at a concentration of about 140 mM, theArg HCl, Arg acetate, or Arg glutamate is present at a concentration ofabout 63 mM, the sodium acetate buffer is present at a concentration ofabout 10 mM. In further sub-aspects, the N-acetyl arginine is present ata concentration of about 155 mM, the Arg HCl, Arg acetate, or Argglutamate is present at a concentration of about 70 mM, the sodiumacetate buffer is present at a concentration of about 10 mM. In yetother sub-aspects, the N-acetyl arginine is present at a concentrationof about 170 mM, the Arg HCl, Arg acetate, or Arg glutamate is presentat a concentration of about 63 mM, the sodium acetate buffer is presentat a concentration of about 10 mM. Furthermore, the compositions canfurther comprise proline, wherein the proline is present at aconcentration of about 50 mM to about 150 mM. The second or thirdsolution can have a pH from about 4.8 to about 6.9, such as 5.4.Furthermore, surfactant can added to the third solution after beingconcentrated, such as a surfactant selected from the group consisting ofpolyoxyethylenesorbitan monooleate (polysorbate 80 or polysorbate 20),polyoxyethylene-polyoxypropylene block copolymer (Poloxamers such asPluronic® F-68 and other Pluronics®), Sorbitan alkyl esters (Spans®)Polyethylene glycol octylphenyl ethers (Triton X-100), Polyethyleneglycol alkyl ethers (Brij), Polypropylene glycol alkyl ethers, Glucosidealkyl ethers, and D-α-tocopherol polyethylene glycol succinate (vitaminE TPGS). The surfactant can be present at a concentration of about0.0001% (w/v) to about 1% (w/v). In some pharmaceutical compositions ofthis aspect, the surfactant is polyoxyethylenesorbitan monooleate(polysorbate 80) and is present at a concentration of about 0.01% (w/v).

In a thirteenth aspect, disclosed herein is a method of formulating atherapeutic polypeptide, comprising

-   a. a first concentration step, wherein the polypeptide in a first    solution is concentrated using fed-batch ultrafiltration;-   b. a first solution exchange step, wherein the concentrated    polypeptide in the first solution is exchanged into a second using    diafiltration and three diavolumes of the second solution;-   c. a second concentration step, wherein the polypeptide in the    second solution is concentrated using fed-batch ultrafiltration;-   d. a second solution exchange step, wherein the polypeptide in the    concentrated second solution is exchanged into third solution using    diafiltration and four diavolumes of the third solution;-   e. the temperature of the solution comprising the polypeptide is    increased from about 25° C. to about 37° C. after the second    solution exchange step; and-   f. a third concentration step, wherein the polypeptide is further    concentrated using fed-batch ultrafiltration concentration;-   g. alternatively, a step adding polyoxyethylenesorbitan monooleate    (polysorbate 80) at a concentration of about 0.01% (w/v) to the    resulting solution of the third concentration step,-   wherein the second and third solutions comprise a solution selected    from the group consisting of a solution comprising about 140 mM    N-acetyl arginine, about 50 mM Arg HCl, and about 10 mM sodium    acetate, the solution having a pH of about 5.2; a solution    comprising about 155 mM N-acetyl arginine, about 70 mM Arg HCl, and    about 10 mM sodium acetate, the solution having a pH of about 5.4;    and a solution comprising about 170 mM N-acetyl arginine, about 10    mM sodium acetate, the solution having a pH of about 5.6;

wherein in the first and second solution exchange steps, a diafiltrationmembrane is used having at least one characteristic selected from thegroup consisting of:

-   a. mesh openings that are greater than about 350 µm but less than or    equal to about 500 µm;-   b. an open area that is greater than about 32% but less than or    equal to about 36% of the membrane area;-   c. a mesh count of less than about 16.2 n/cm but greater than or    equal to about 12.2 n/cm;-   d. a wire diameter that is greater than about 270 µm but less than    or equal to about 340 µm;-   e. a basis weight that is greater than about 160 g/m² but less than    or equal to 180 g/m²;-   f. a thickness greater than about 515 µm but less than or equal to    about 610 µm;-   g. a membrane load of greater than about 1138.1 g/m² but less than    or equal to about 1919.3 g/m²; and-   h. a maximum feed pressure of about 60 psi; and

wherein the therapeutic polypeptide comprises a PCSK9-bindingpolypeptide that blocks binding of PCSK9 to LDLR and is selected fromthe group consisting of:

-   i. a monoclonal antibody comprising a heavy chain having an amino    acid sequence of SEQ ID NO:1 and a light chain having an amino acid    sequence of SEQ ID NO:2 (evolocumab), or an antigen-binding fragment    thereof;-   ii. a monoclonal antibody that competes with evolocumab for binding    to PCSK9;-   iii. a monoclonal antibody, comprising:    -   1. a heavy chain polypeptide comprising the following        complementarity determining regions (CDRs): a heavy chain CDR1        that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain CDR2 that        is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain CDR3 that is a        CDR3 in SEQ ID NOs:14 or 16, and    -   2. a light chain polypeptide comprising the following CDRs: a        light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a light        chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a light chain        CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;-   iv. a monoclonal antibody that binds to at least one of the    following residues of PCSK9, the PCSK9 comprising an amino acid    sequence of SEQ ID NO:3: S153, D188, l189, Q190, S191, D192, R194,    E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376,    T377, F379, l154, T1897, H193, E195, l196, M201, V202, C223, T228,    S235, G236, A239, G244, M247, l369, S372, C375, C378, R237, D238;-   v. a monoclonal antibody that binds to PCSK9 at an epitope on PCSK9    that overlaps with an epitope that is bound by an antibody that    comprises:    -   1. a heavy chain variable region of the amino acid sequence in        SEQ ID NO:1; and    -   2. a light chain variable region of the amino acid sequence in        SEQ ID NO:2, and    -   3. wherein the epitope of the monoclonal antibody further        overlaps with a site to which an EGFa domain of LDLR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph of the data from size-exclusion chromatography-highpressure liquid chromatography (SE-HPLC) of evolocumab samples at highconcentrations in various formulations containing N-acetyl argininefollowing 1 month incubation at 40° C. Key to formulations indicated onx-axis: [acetate (mM)]/[N-acetyl arginine (mM)]/[proline (mM)]. Y-axisindicates percentage of sample that is high molecular weight (HMW)species (aggregates and oligomers of evolocumab).

FIG. 2 shows a graph of a formulation viscosity comparison of evolocumabat 210 mg/mL -1000 sec⁻¹ shear rate at 25° C.

FIG. 3A shows graphs produced by JMP Prediction Profiler software ofSE-HPLC data for evolocumab formulation held at 40° C. for one month.FIG. 3B shows similar data, except the samples were held at 25° C. forthree months.

FIG. 4 shows a bar graph of the percent HMW species by SE-HPLC of 210mg/mL evolocumab formulations held at 40° C. for one month. Formulationsvary by concentrations of N-acetyl arginine and arginine HClconcentration at varying pH. FIGS. 5A-5C show SE-HPLC chromatographs ofselected pH 4.8 and pH 5.4 evolocumab samples held at the indicatedtemperatures and times (5° C. for six months (FIG. 5A); 25° C. for threemonths (FIG. 5B); and 40° C. for one month (FIG. 5C)) and compared to aevolocumab proline formulation control. Key to formulations: [acetate(mM)]/[N-acetyl arginine (mM)]/[arginine HCl (mM)].

FIGS. 6A and 6B shows graphs of the percent of HMW species that developover time for the indicated evolocumab formulations held at theindicated temperatures (5° C. (FIG. 6A); 25° C. (FIG. 6B)). The testedevolocumab N-acetyl arginine formulations at 210 mg/mL are compared toan evolocumab proline formulation control at 140 mg/mL. Key toformulations: [acetate (mM)]/[N-acetyl arginine (mM)]/[arginine HCl(mM)].

FIG. 7A shows graphs produced by JMP Prediction Profiler software ofcation exchange chromatography-high pressure liquid chromatography(CEX-HPLC) data for evolocumab formulations held at 40° C. for onemonth. FIG. 7B shows similar data, except the samples were held at 25°C. for three months. See the Examples for further details.

FIG. 8 shows a graph of the percent acidic peak by CEX-HPLC of 210 mg/mLevolocumab formulations held at 40° C. for one month.

FIGS. 9A-9B show graphs of sub-visible particles as determined by lightobscuration liquid borne particle counting (equal to or greater than 10µM, FIG. 9A; or equal to or greater than 25 µM, FIG. 9B) per milliliterof various evolocumab formulations held at 5° C., 25° C., and 40° C. forthree months.

FIG. 9C shows a graph of sub-visible particles as determined by lightobscuration liquid borne particle counting (greater than or equal to 10µM or 25 µM) of various evolocumab formulations held at 5° C. or 25° C.for six months.

FIG. 10 shows a graph of sub-visible particles of various evolocumabformulations as determined by Micro Flow Imaging (MFI) analysis,filtered by aspect ratio (AR) of less than 0.70.

FIG. 11 shows a summary graph of the viscosities of various evolocumabformulations held for the specified times and temperatures as indicatedwhere T0 represents the initial viscosity values.

FIG. 12 shows a graph and a table showing the pH values of evolocumabformulations differing from each other by pH and buffer over time, up tothree months. The samples were held at 40° C.

FIGS. 13A-13C show graphs of SE-HPLC data that demonstrate the effect ofpH on percent HMW species of 210 mg/mL evolocumab formulations thatdiffer by pH and buffer for up to three months at 4° C. (FIG. 13A), 25°C. (FIG. 13B), and 40° C. (FIG. 13C). In addition to 10 mM of eachlisted buffer, samples all contain 165 mM N-acetyl arginine, 75 mMarginine HCl, and 0.01% polysorbate-80.

FIG. 14 shows a graph of oligomer levels of evolocumab formulations atvarying pH immediately after formulation.

FIG. 15 shows chromatograms showing various sizes of protein moleculesof evolocumab formulations that differ by pH and buffer after being heldat 40° C. for three months. (LMW = low molecular weight species).

FIGS. 16A-16C show graphs of data from CEX-HPLC analyses (% main peak)of 210 mg/mL evolocumab formulations that differ by pH and buffer overtime. FIG. 16A shows percent main peak for samples held at 5° C. overtime, while FIG. 16B shows the same type of data for samples held at 25°C. and FIG. 16C shows the same type of data for samples held at 40° C.In addition to 10 mM of each listed buffer, samples all contain 165 mMN-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80.

FIG. 17 shows a graph of data from CEX-HPLC analyses of 210 mg/mLevolocumab formulations that differ by pH and buffer held at 40° C. overtime, measuring percent of acidic species in the formulations. Inaddition to 10 mM of each listed buffer, samples all contain 165 mMN-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80.

FIG. 18 shows chromatograms of CEX-HPLC data of 210 mg/mL evolocumabformulations that differ by pH and buffer held at 25° C. for threemonths, analyzing showing acidic, and basic, and main peak species. Inaddition to 10 mM of each listed buffer, samples all contain 165 mMN-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80.

FIG. 19 shows chromatograms of peptide mapping data of variousevolocumab formulations differing by pH and buffer, the samples havingbeen held at 40° C. for one month.. In addition to 10 mM of each listedbuffer, samples all contain 165 mM N-acetyl arginine, 75 mM arginineHCl, and 0.01% polysorbate-80.

FIG. 20 shows a graph summarizing the results of several experimentsthat analyzed evolocumab in formulations that differ by pH and bufferafter 1 month at 40° C. All samples contain lOmM buffer 165 mM N-acetylarginine, 75 mM arginine HCl, and 0.01% polysorbate-80. Refer to thefigure and the Examples for further details.

FIGS. 21A-21B show graphs of sub-visible particles as determined bylight obscuration liquid borne particle counting (equal to or greaterthan 10 µM, FIG. 21A; or equal to or greater than 25 µM, FIG. 21B) permilliliter of various evolocumab formulations differing by pH and bufferheld at 5° C., 25° C., and 40° C. for two months. FIGS. 21C-21D showgraphs of sub-visible particles (greater than or equal to 10 µM, FIG.21C; greater than or equal to 25 µM, FIG. 21D) of evolocumabformulations differing by pH and buffer held at 5° C. or 25° C. for sixmonths. In addition to lOmM of each listed buffer, samples all contain165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80.

FIG. 22 shows a graph of sub-visible particles of evolocumabformulations that differ by pH and buffer as ascertained by Micro FlowImaging (MFI). In addition to 10 mM of each listed buffer, samples allcontain 165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01%polysorbate-80.

FIG. 23 shows a graph of the results of reduced capillaryelectrophoresis-sodium dodecyl sulfate (rCE-SDS) analyses on 210 mg/mLevolocumab formulations varying by pH and buffer held at 25° C. for 6months. In addition to 10 mM of each listed buffer, samples all contain165 mM N-acetyl arginine, 75 mM arginine HCl, and 0.01% polysorbate-80.

FIG. 24 shows a graph of the relationship between viscosity and pH ofevolocumab formulations that differ in pH and buffer. All samplescontain 10 mM buffer 165 mM N-acetyl arginine, 75 mM arginine HCl, and0.01% polysorbate-80.

FIGS. 25A-25C show graphs of percent HMW species as measured by SE-HPLCof different evolocumab formulations held for the indicated time periodsat 5° C. (FIG. 25A), 25° C. (FIG. 25B), and 40° C. (FIG. 25C). Key toformulations: [acetate (mM)]/[N-acetyl arginine (mM)]/[arginine HCl(mM)].

FIGS. 26A-26C show graphs of percent acidic peak as measured by CEX-HPLCof different evolocumab formulations held for the indicated time periodsat 5° C. (FIG. 26A), 25° C. (FIG. 26B), and 40° C. (FIG. 26C). Key toformulations: [acetate (mM)]/[N-acetyl arginine (mM)]/[arginine HCl(mM)].

FIG. 27 shows a graph of percent Pre -LC + LC + HC (LC = Light chain, HC= Heavy Chain) by reduced capillary electrophoresis-sodium dodecylsulfate (rCE-SDS) of different evolocumab formulations held for threemonths at 5° C., 25° C., and 40° C. compared to initial levels (T0) .Key to formulations: [acetate (mM)]/[N-acetyl arginine (mM)]/[arginineHCl (mM)].

FIG. 28 shows a graph of the viscosities of different evolocumabformulations of three different concentrations of evolocumab; theviscosity data was determined using a rheometer at shear rates up to90,000 sec⁻¹ at the indicated temperatures. Key to formulations:[acetate (mM)]/[N-acetyl arginine (mM)]/[arginine HCl (mM)].

FIG. 29 shows a graph of evolocumab UF/DF flux data in three NARformulation buffers.

FIG. 30 shows a graph of the percentage of HMW species formation in theevolocumab UF/DF process with 35 mg/mL and 70 mg/mL evolocumab inUF1/DF1 (UF/DF-70 and UF/DF-35). The figure also shows the concentrationof evolocumab in mg/mL at each step of the process for the two initialconcentrations of evolocumab.

FIGS. 31A-31F show graphs of SE-HPLC (FIGS. 31A, 31C, and 31E) andCEX-HPLC (FIGS. 31B, 31D, and 31F) of evolocumab in NAR-containingformulations at pH 5.2 (FIGS. 31A and 31B), pH 5.4 (FIGS. 31C and 31D),and pH 5.6 (FIGS. 31E and 31F). The values of the bars for the percentHMW species, percent LMW species, and percent evolocumab shown in FIGS.31A-31F are provided in Tables 17A-17F.

FIG. 32 shows percent HMW species formation of evolocumab NAR DS samplefrom pool hold studies at 2-8° C. and room temperature.

FIG. 33 shows a graph of percent HMW species formation of evolocumab NAROC samples from pool hold studies at elevated temperature

FIG. 34 shows a graph of viscosity measurements of evolocumab NARformulations at different temperatures.

FIGS. 35A-35C show graphs of SE-HPLC data for all of the studyformulations used in Example 9 following up to 6 months incubation at 4°C. (FIG. 35A), 25° C. (FIG. 35B), and 40° C. (FIG. 35C). FIG. 35Ddisplays the 40° C. SE-HPLC data (shown as a line graph in FIG. 35C) asa bar chart, making the comparison of aggregation levels betweenformulations more discernable.

FIGS. 36A-36F show graphs of CEX-HPLC data for the study formulationsused in Example 9 showing changes in % acidic and % basic peaks overtime following up to three months incubation at 4° C. (FIG. 36A (%acidic), FIG. 36B (% basic)), 25° C. (FIG. 36C (% acidic), FIG. 36D (%basic)), and 40° C. (FIG. 36E (% acidic), FIG. 36F (% basic)).

FIGS. 37A-37D show graphs of rCE-SDS data for % main peak and % LMWspecies for the study formulations used in Example 9 over time followingup to three months incubation at 30° C. (FIG. 37A (% main peak), FIG.37B (% LMW species)) and 40° C. (FIG. 37C (% main peak), FIG. 37D (% LMWspecies)).

FIGS. 38A-38D show graphs of subvisible particle data by lightobscuration particle counting using HIAC for the study formulations usedin Example 9 following incubation at 4° C. and 40° C. for up to threemonths. FIG. 38A: HIAC — ≥ 10 µm - 4° C.; FIG. 38B: HIAC — ≥ 25 µm - 4°C.; FIG. 38C: HIAC — ≥ 10 µm - 40° C.; and HlAC — ≥ 25 µm - 40° C.

DETAILED DESCRIPTION

The inventors have surprisingly discovered that a derivative ofarginine, N-acetyl arginine (NAR), efficiently reduces the viscosity ofpharmaceutical compositions comprising high concentrations (greater than100 mg/mL, such as 140 mg/mL and greater) of evolocumab more so thanunacetylated arginine. Pharmaceutical compositions having a viscosity of50 cP or less are amenable to manufacturing and patient administrationwithout significant complications, while higher viscosity preparationsare difficult to handle (such as when syringes are filled), and toadminister. While unmodified arginine is known to reduce viscosity ofhigh protein concentration formulations, arginine glutamate only reducedthe viscosity of a comparable proline-containing evolocumab (210 mg/mL)formulation by 50 cP (from 159 cP to 109 cP), substantially higher thana target of 50 cP or less. Furthermore, arginine monohydrochloride (ArgHCl) addition alone did not achieve this goal, reducing viscosity from159 cP of the proline-containing formulation to 70 cP. However, NARachieved a surprising effect of further reducing the viscosity to 58cP - a reduction of over 101 cP relative to a proline formulation, andwhen combined with Arg HCl (to increase the solubility of NAR andachieve an isotonic formulation), a viscosity of under 50 cP wasachieved; in fact, the goal was exceeded by 7 cP, the formulation havinga viscosity of 43 cP. See FIG. 2 . Since NAR solubility is limited toless than 230 mM, another excipient is necessary to achieve an isotonicformulation for subcutaneous administration. The unexpected discoverythat the chloride salt of arginine is more effective at reducingviscosity of evolocumab than other arginine salts such as glutamate wascritical to minimizing formulation viscosity.

Another unexpected discovery was the pH dependent evolocumab stabilityand viscosity effects seen in the presence of NAR and arginine HCl. Asignificant increase in the rate of evolocumab aggregation was seen atpH less than 5.0 at elevated temperatures. Also, a pH dependent decreasein viscosity was observed as pH increased from pH 5.1 to 6.9.

The inventors further discovered that a two-stepultrafiltration/diafiltration (UF/DF) process can prepare suchNAR-containing, high evolocumab concentration pharmaceuticalformulations with a significant savings in NAR material compared totraditional one-step traditional processes. Such methods allow forsignificant cost savings, as NAR is about ten-fold more expensive thanArg HCl.

Definitions

Both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictive.The use of the singular includes the plural unless specifically statedotherwise. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including”, as well as other forms, such as “includes”and “included”, is not limiting. Terms such as “element” or “component”encompass both elements and components comprising one unit and elementsand components that comprise more than one subunit unless specificallystated otherwise. The use of the term “portion” can include part of amoiety or the entire moiety. When a numerical range is mentioned, e.g.,1-5, all intervening values are explicitly included, such as 1, 2, 3, 4,and 5, as well as fractions thereof, such as 1.5, 2.2, 3.4, and 4.1.

“About” or “^(∼)” mean, when modifying a quantity (e.g., “about” 3 mM),that variation around the modified quantity can occur. These variationscan occur by a variety of means, such as typical measuring and handlingprocedures, inadvertent errors, ingredient purity, and the like.

“N-acetyl arginine” (NAR) means the molecule of formula 1.

“Additive” means, in the context of a pharmaceutical composition, asubstance not naturally part of a material (e.g., drug substance) butdeliberately added to fulfill some specific purpose (e.g., preservation,viscosity reduction, stabilization).

“Analog” refers to an amino acid sequence that has insertions, deletionsor substitutions relative to the parent sequence, while stillsubstantially maintaining the biological activity of the parentsequence, as determined using biological assays. Analogs includepolypeptides with modified glycosylation, polypeptides withoutglycosylation. Formulations can also include derivatives of naturallyoccurring or analog polypeptides which have been chemically modified,for example, to attach water soluble polymers (e.g., PEGylated),radionuclides, or other diagnostic or targeting or therapeutic moieties.

“Antibody” refers to an intact immunoglobulin of any isotype, andincludes, for instance, chimeric, humanized, human, and bispecificantibodies. An intact antibody will generally comprise at least twofull-length heavy chains and two full-length light chains. Antibodysequences can be derived solely from a single species, or can be“chimeric,” that is, different portions of the antibody can be derivedfrom two different species. “Antibody” also includes antibodiescomprising two substantially full-length heavy chains and twosubstantially full-length light chains provided the antibodies retainthe same or similar binding and/or function as the antibody comprised oftwo full length light and heavy chains. For example, antibodies having1, 2, 3, 4, or 5 amino acid residue substitutions, insertions ordeletions at the N-terminus and/or C-terminus of the heavy and/or lightchains are included in the definition provided that the antibodiesretain the same or similar binding and/or function as the antibodiescomprising two full length heavy chains and two full length lightchains. Antibodies include, for example, monoclonal antibodies,polyclonal antibodies, chimeric antibodies, humanized antibodies, humanantibodies, bispecific antibodies, and synthetic antibodies.

Typical antibody structural units comprise a tetramer. Each suchtetramer typically is composed of two identical pairs of polypeptidechains, each pair having one full-length “light” (about 25 kDa) and onefull-length “heavy” chain (about 50-70 kDa). The amino-terminal portionof each chain typically includes a variable region of about 100 to 110or more amino acids that typically is responsible for antigenrecognition. The carboxy-terminal portion of each chain typicallydefines a constant region that can be responsible for effector function.Light chains are typically classified as kappa and lambda light chains.Heavy chains are typically classified as mu, delta, gamma, alpha, orepsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, andIgE, respectively. IgG antibodies have several subclasses, includingIgG1, IgG2, IgG3, and IgG4. IgM has subclasses including IgM1 and IgM2.IgA is similarly subdivided into subclasses including IgA1 and IgA2.Within full-length light and heavy chains, typically, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about tenmore amino acids. The variable regions of each light/heavy chain pairtypically form the antigen binding site.

The variable regions typically exhibit the same general structure ofrelatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions orCDRs. The CDRs from the two chains of each pair typically are aligned bythe framework regions, which can enable binding to a specific epitope.From N-terminal to C-terminal, both light and heavy chain variableregions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is typically inaccordance with the definitions of Kabat (Kabat, Wu, Perry, Gottesman, &Foeller, 1991; Kabat, Wu, Reid-Miller, Perry, & Gottesman, 1987) orChothia (Chothia & Lesk, 1987; Chothia et al., 1989).

Instead of a full length antibody, a “fragment” or “antigen bindingfragment” of an antibody can be used. An “antibody fragment” refers tothe Fab, Fab‘, F(ab‘)2, and Fv fragments that contain at least one CDRof an immunoglobulin that is sufficient to confer specific antigenbinding to the target protein, such as PCSK9.

An antibody heavy chain can bind to an antigen in the absence of anantibody light chain. An antibody light chain can bind to an antigen inthe absence of an antibody heavy chain. An antibody binding region canbind to an antigen in the absence of an antibody light chain. Anantibody binding region can bind to an antigen in the absence of anantibody heavy chain. An individual variable region can specificallybind to an antigen in the absence of other variable regions.

The CDR regions in the heavy chain are typically referred to as H1, H2,and H3 and are numbered sequentially in the direction from the aminoterminus to the carboxy terminus. The CDR regions in the light chain arereferred to as L1, L2, and L3 and are numbered sequentially in thedirection from the amino terminus to the carboxy terminus.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length light chain includes a variable regiondomain, VL, and a constant region domain, CL. The variable region domainof the light chain is at the amino-terminus of the polypeptide. Lightchains include kappa chains and lambda chains.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length heavy chain includes a variable regiondomain, VH, and three constant region domains, CH1, CH2, and CH3. The VHdomain is at the amino-terminus of the polypeptide, and the CH domainsare at the carboxyl-terminus, with the CH3 being closest to thecarboxy-terminus of the polypeptide. Heavy chains can be of any isotype,including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA(including IgA1 and IgA2 subtypes), IgM and IgE.

Each individual immunoglobulin chain is typically composed of several“immunoglobulin domains,” each consisting of roughly 90 to 110 aminoacids and having a characteristic folding pattern. These domains are thebasic units of antibody polypeptides. In humans, the IgA and IgDisotypes contain four heavy chains and four light chains; the IgG andIgE isotypes contain two heavy chains and two light chains; and the IgMisotype contains five heavy chains and five light chains. The heavychain C region typically comprises one or more domains that can beresponsible for effector function. The number of heavy chain constantregion domains depend on the isotype. IgG heavy chains, for example,contain three C region domains known as CH1, CH2 and CH3. In certaincases, an anti-PCSK9 antibody is an IgG1 or IgG2 or IgG4 subtype.

The term “variable region” or “variable domain” refers to a portion ofthe light and/or heavy chains of an antibody, typically includingapproximately the amino-terminal 120 to 130 amino acids in the heavychain and about 100 to 110 amino terminal amino acids in the lightchain. The variable region of an antibody typically determinesspecificity of a particular antibody for its target.

“Antigen” means a molecule or a portion of a molecule capable of beingbound by a selective binding agent, such as a PCSK9-binding polypeptide(including, e.g., an antibody or binding fragment thereof). In somecases, the antigen is capable of being used in an animal to produceantibodies capable of binding to that antigen. An antigen can possessone or more epitopes that are capable of interacting with differentPCSK9-binding polypeptides.

“Arginine salt” means a salt of arginine. Examples include argininemonohydrochloride (Arg HCl), arginine acetate (Arg acetate) and arginineglutamate (Arg glutamate).

“Buffer” means any pharmaceutically acceptable buffer, includingacetate, glutamate, histidine, and phosphate buffers, and salts thereof.

“Compete” when used in the context of antibodies that compete for thesame epitope means competition between antibodies as determined by anassay in which the antibodies being tested prevents or inhibits (e.g.,reduces) specific binding of a reference antibody (e.g., a ligand, or areference antibody) to a common antigen (e.g., PCSK9 or a fragmentthereof). Numerous types of competitive binding assays can be used todetermine if one antibody competes with another, for example: solidphase direct or indirect immunoassays using a variety or art-acceptedreagents and labels. Competitive inhibition is measured by determiningthe amount of label bound to the solid surface or cells in the presenceof the test antibody. Usually the test antibody is present in excess.Antibodies identified by competition assay include antibodies binding tothe same epitope as the reference antibody and antibodies binding to anadjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur. Usually, when acompeting antibody is present in excess, it will inhibit (e.g., reduce)specific binding of a reference antibody to a common antigen by at least40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or 75% or more.In some instances, binding is inhibited by at least 80-85%, 85-90%,90-95%, 95-97%, or 97% or more.

“Diafiltration,” “DF,” and like terms mean using an ultrafiltrationmembrane (i.e., a semipermeable membrane that can discriminate betweenmolecules having different shapes and sizes) to remove, replace, orlower the concentration of salts or solvents from solutions or mixturescontaining, for example, polypeptides or other biomolecules.

“Diavolume (DV)” means, in the context of filtration, the volume ofdiafiltration buffer introduced into the unit operation compared to theretentate volume.

“Epitope” includes any determinant capable of being bound by aPCSK9-binding polypeptide, such as an antibody. An epitope is a regionof an antigen that is bound by a PCSK9-binding polypeptide that targetsthat antigen, and when the antigen is a protein, includes specific aminoacids that directly contact the PCSK9-binding polypeptide. Epitopedeterminants can include chemically active surface groupings ofmolecules such as amino acids, sugar side chains, phosphoryl groups andsulfonyl groups and can have specific three dimensional structuralcharacteristics and specific charge characteristics. Generally,antibodies specific for a particular target antigen preferentiallyrecognize an epitope on the target antigen in a complex mixture ofproteins or other macromolecules.

“Excipient” means more or less an inert substance added in aprescription as a diluent or vehicle or to give form or consistency whenthe remedy is given in pill form; e.g., simple syrup, vegetable gums,aromatic powder, honey, and various elixirs.

“Feed Cross-flow” means the feed flow rate (L/hour) divided by membranearea (m²).

“Flux (LMH)” means, in the context of filtration, liters per hour persquare meter of membrane area (L/h/m²).

“High molecular weight species” or “HMW species” means, in the contextof a pharmaceutical formulation containing a therapeutic polypeptide,therapeutic proteins that are larger than the original therapeuticpolypeptide, as determined by art-accepted assays. HMW species includeoligomers of therapeutic polypeptides and aggregates of therapeuticpolypeptides.

“Holdup Volume (HUV)” means, in the context of filtration, the linevolume of the TFF system, including that of the cartridge.

“Identity” refers to a relationship between the sequences of two or morepolypeptide molecules or two or more nucleic acid molecules, asdetermined by aligning and comparing the sequences. “Percent identity”means the percent of identical residues between the amino acids ornucleotides in the compared molecules and is calculated based on thesize of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) are preferably addressed by aparticular mathematical model or algorithm. These techniques arewell-known in the art.

In calculating percent identity, the sequences being compared aretypically aligned to maximize the largest match between the sequences.

Certain alignment schemes for aligning two amino acid sequences canresult in matching only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method can be adjusted ifso desired to result in an alignment that spans a desired number ofcontiguous amino acids (e.g., 50 amino acids) of the target polypeptide.

Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidscan also be suitable components for PCSK9-binding polypeptides. Examplesof unconventional amino acids include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids (e.g., 4-hydroxyproline).

Conservative amino acid substitutions can encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

In making changes to the antigen binding protein or the PCSK9 protein,the hydropathic index of amino acids can be considered. Each amino acidhas been assigned a hydropathic index on the basis of its hydrophobicityand charge characteristics. The importance of the hydropathic amino acidindex in conferring interactive biological function on a protein isunderstood in the art. Certain amino acids can be substituted for otheramino acids having a similar hydropathic index or score and still retaina similar biological activity.

The substitution of like amino acids can be made effectively on thebasis of hydrophilicity. One can also identify epitopes from primaryamino acid sequences on the basis of hydrophilicity. These regions arealso referred to as “epitopic core regions.”

“Low molecular weight species” or “LMW species” means, in the context ofa pharmaceutical formulation containing a therapeutic polypeptide,polypeptides that are smaller than the original therapeutic polypeptide,as determined by art-accepted assays. LMW species include fragments ofthe therapeutic polypeptide.

“Neutralizing antibody” or an “antibody that neutralizes a target” asused in “anti-PCSK9 neutralizing antibody” refers to an antibody thatbinds to a target and prevents or reduces the biological activity ofthat target. This can be done, for example, by directly blocking abinding site on the target or by binding to the target and altering thetarget’s ability to bind through indirect means, such as structural orenergetic alterations in the target. In assessing the binding and/orspecificity of an antibody or immunologically functional fragmentthereof, an antibody or fragment can substantially inhibit binding of atarget to its binding partner when an excess of antibody reduces thequantity of binding partner bound to the ligand by at least about 1-20%,20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%,95-97%, 97-98%, 98-99% or more (as measured in an in vitro competitivebinding assay). In the case of PCSK9 antibodies, such a neutralizingmolecule can diminish the ability of PCSK9 to bind the LDLR. In somecases, the neutralizing ability is characterized or described via acompetition assay. In some cases, the neutralizing ability is describedin terms of an IC50 or EC50 value. In some cases, the antibodiesneutralize by binding to PCSK9 and preventing PCSK9 from binding toLDLR, or reducing the ability of PCSK9 to bind to LDLR. In some cases,the antibodies neutralize by binding to PCSK9, while still allowingPCSK9 to bind to LDLR, preventing or reducing the PCSK9 mediateddegradation of LDLR. Thus, in some instances, a neutralizing antibodycan still permit PCSK9/LDLR binding, but prevents or reduces subsequentPCSK9 involved degradation of LDLR. Neutralizing results in the loweringLDL-C (and/or other lipids, such as ApoB, Lp(a), etc.). PCSK9-bindingpolypeptides beyond antibodies, including variants of such PCSK-bindingpolypeptides, can have these same activities.

“PCSK9-binding polypeptide” means a polypeptide that binds proproteinconvertase subtilisin/kexin type 9 (PCSK9) protein. In some cases, thePCSK9-binding polypeptide blocks binding of PCSK9 to low-density lipidreceptors (LDLRs). Such blocking PCSK9-binding polypeptides can bemonoclonal antibodies (mAbs) and can be one of the following:

-   a. a mAb comprising a heavy chain polypeptide having an amino acid    sequence of SEQ ID NO:1 and a light chain polypeptide having an    amino acid sequence of SEQ ID NO:2 (evolocumab), or an    antigen-binding fragment thereof;-   b. a mAb that competes with evolocumab for binding to PCSK9;-   c. a mAb, comprising:    -   i. a heavy chain polypeptide comprising the following        complementarity determining regions (CDRs): a heavy chain CDR1        that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain CDR2 that        is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain CDR3 that is a        CDR3 in SEQ ID NOs:14 or 16, and    -   ii. a light chain polypeptide comprising the following CDRs: a        light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a light        chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a light chain        CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;-   d. a mAb that binds to at least one of the following residues of    PCSK9, the PCSK9 comprising an amino acid sequence of SEQ ID NO:3:    S153, D188, I189, Q190, S191, D192, R194, E197, G198, R199, V200,    D224, R237, and D238, K243, S373, D374, S376, T377, F379, I154,    T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239,    G244, M247, I369, S372, C375, C378, R237, and D238;-   e. a mAb that binds to PCSK9 at an epitope on PCSK9 that overlaps    with an epitope that is bound by an antibody that comprises:    -   i. a heavy chain variable region of the amino acid sequence in        SEQ ID NO:1; and    -   ii. a light chain variable region of the amino acid sequence in        SEQ ID NO:2, and    -   iii. wherein the epitope of the mAb further overlaps with a site        to which an epidermal growth factor-like repeat A (EGF-A) domain        of LDLR; or-   f. a mAb the comprises a heavy chain polypeptide comprising the    following complementarity determining regions (CDRs):    -   i. heavy chain CDR1, CDR2, and CDR3 having an amino acid        sequence of SEQ ID NOs:7, 8, and 9, respectively; and    -   ii. light chain CDR1, CDR2, and CDR3 having an amino acid        sequence of SEQ ID NOs:4, 5, and 6, respectively.

The indicated amino acid sequences are presented in Table 1, which alsopresents the heavy chain variable region and light chain variable regionof evolocumab. Evolocumab heavy chain and light chain full-lengthnucleotide sequences are given in Table 2, as are the nucleotidesequences for the evolocumab HCVR and LCVR.

TABLE 1 PCSK9 and PCSK9-binding polypeptide sequences Evolocumab HCsequence (USAN; SEQ ID NO:1)EVQLVQSGAE VKKPGASVKV SCKASGYTLT SYGISWVRQA PGQGLEWMGW VSFYNGNTNY 60AQKLQGRGTM TTDPSTSTAY MELRSLRSDD TAVYYCARGY GMDVWGQGTT VTVSSASTKG 120PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL 180SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE CPPCPAPPVA GPSVFLFPPK 240PKDTLMISRT PEVTCVVVDV SHEDPEVQFN WYVDGVEVHN AKTKPREEQF NSTFRVVSVL 300TVVHQDWLNG KEYKCKVSNK GLPAPIEKTI SKTKGQPREP QVYTLPPSRE EMTKNQVSLT 360CLVKGFYPSD IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR WQQGNVFSCS 420VMHEALHNHY TQKSLSLSPG K 441 Evolocumab LC sequence (USAN; SEQ ID NO:2)ESALTQPASV SGSPGQSITI SCTGTSSDVG GYNSVSWYQQ HPGKAPKLMI YEVSNRPSGV 60SNRFSGSKSG NTASLTISGL QAEDEADYYC NSYTSTSMVF GGGTKLTVLG QPKAAPSVTL 120FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTTPSKQ SNNKYAASSY 180LSLTPEQWKS HRSYSCQVTH EGSTVEKTVA PTECS 215 PCSK9 preproprotein (human;SEQ ID NO:3)MGTVSSRRSW WPLPLLLLLL LLLGPAGARA QEDEDGDYEE LVLALRSEED GLAEAPEHGT 60TATFHRCAKD PWRLPGTYVV VLKEETHLSQ SERTARRLQA QAARRGYLTK ILHVFHGLLP 120GFLVKMSGDL LELALKLPHV DYIEEDSSVF AQSIPWNLER ITPPRYRADE YQPPDGGSLV 180EVYLLDTSIQ SDHREIEGRV MVTDFENVPE EDGTRFHRQA SKCDSHGTHL AGVVSGRDAG 240VAKGASMRSL RVLNCQGKGT VSGTLIGLEF IRKSQLVQPV GPLVVLLPLA GGYSRVLNAA 300CQRLARAGVV LVTAAGNFRD DACLYSPASA PEVITVGATN AQDQPVTLGT LGTNFGRCVD 360LFAPGEDIIG ASSDCSTCFV SQSGTSQAAA HVAGIAAMML SAEPELTLAE LRQRLIHFSA 420KDVINEAWFP EDQRVLTPNL VAALPPSTHG AGWQLFCRTV WSAHSGPTRM ATAIARCAPD 480EELLSCSSFS RSGKRRGERM EAQGGKLVCR AHNAFGGEGV YAIARCCLLP QANCSVHTAP 540PAEASMGTRV HCHQQGHVLT GCSSHWEVED LGTHKPPVLR PRGQPNQCVG HREASIHASC 600CHAPGLECKV KEHGIPAPQG QVTVACEEGW TLTGCSALPG TSHVLGAYAV DNTCVVRSRD 660VSTTGSTSEE AVTAVAICCR SRHLAQASQE LQ 692 Evolocumab HCVR sequence (SEQ IDNO:14)QVQLVQSGAE VKKPGASVKV SCKASGYTLT SYGISWVRQA PGQGLEWMGW VSFYNGNTNY 60AQKLQGRGTM TTDPSTSTAY MELRSLRSDD TAVYYCARGY GMDVWGQGTT VTVSS 115Evolocumab LCVR sequence (SEQ ID NO:15)QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNSVSWYQQ HPGKAPKLMI YEVSNRPSGV  60SNRFSGSKSG NTASLTISGL QAEDEADYYC NSYTSTSMVF GGGTKLTVL 109 LC CDR1 (SEQID NO:4) TGTSSDVGGY NSVS 14 LC CDR2 (SEQ ID NO:5) EVSNRPS 7 LC CDR3 (SEQID NO:6) NSYTSTSMV 9 HC CDR1 (SEQ ID NO:7) GYTLTSYGIS 10 HC CDR2 (SEQ IDNO:8) WVSFYNGNTN YAQKLQ 16 HC CDR3 (SEQ ID NO:9) GYGMDV 6

TABLE 2 Evolocumab polynucleotide sequences Evolocumab HC sequence (SEQID NO:12) (Note that nucleotides 1 to 57 encode the native signalpeptide)atggactgga cctggaggat ccttttcttg gtggcagcag ccacaggtgt ccactccgag 60gttcagctgg tgcagtctgg agctgaggtg aagaagcctg gggcctcagt gaaggtctcc 120tgcaaggctt ctggttacac cttaaccagc tatggtatca gctgggtgcg acaggcccct 180ggacaagggc ttgagtggat gggatgggtc agtttttata atggtaacac aaactatgca 240cagaagctcc agggcagagg caccatgacc acagacccat ccacgagcac agcctacatg 300gagctgagga gcctgagatc tgacgacacg gccgtgtatt actgtgcgag aggctacggt 360atggacgtct ggggccaagg gaccacggtc accgtctcct ctgcctccac caagggccca 420tcggtcttcc ccctggcgcc ctgctccagg agcacctccg agagcacagc ggccctgggc 480tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc aggcgctctg 540accagcggcg tgcacacctt cccagctgtc ctacagtcct caggactcta ctccctcagc 600agcgtggtga ccgtgccctc cagcaacttc ggcacccaga cctacacctg caacgtagat 660cacaagccca gcaacaccaa ggtggacaag acagttgagc gcaaatgttg tgtcgagtgc 720ccaccgtgcc cagcaccacc tgtggcagga ccgtcagtct tcctcttccc cccaaaaccc 780aaggacaccc tcatgatctc ccggacccct gaggtcacgt gcgtggtggt ggacgtgagc 840cacgaagacc ccgaggtcca gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 900aagacaaagc cacgggagga gcagttcaac agcacgttcc gtgtggtcag cgtcctcacc 960gttgtgcacc aggactggct gaacggcaag gagtacaagt gcaaggtctc caacaaaggc 1020ctcccagccc ccatcgagaa aaccatctcc aaaaccaaag ggcagccccg agaaccacag 1080gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc 1140ctggtcaaag gcttctaccc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1200gagaacaact acaagaccac acctcccatg ctggactccg acggctcctt cttcctctac 1260agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1320atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa 1380Evolocumab LC sequence (SEQ ID NO:13)atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcc 60agatgtgagt ctgccctgac tcagcctgcc tccgtgtctg ggtctcctgg acagtcgatc 120accatctcct gcactggaac cagcagtgac gttggtggtt ataactctgt ctcctggtac 180caacagcacc caggcaaagc ccccaaactc atgatttatg aggtcagtaa tcggccctca 240ggggtttcta atcgcttctc tggctccaag tctggcaaca cggcctccct gaccatctct 300gggctccagg ctgaggacga ggctgattat tactgcaatt catatacaag caccagcatg 360actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 480aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 600agctatctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 660acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc a 711 EvolocumabHCVR sequence (SEQ ID NO:10)caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtc 60tcctgcaagg cttctggtta caccttaacc agctatggta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg gtcagttttt ataatggtaa cacaaactat 180gcacagaagc tccagggcag aggcaccatg accacagacc catccacgag cacagcctac 240atggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagaggctac 300ggtatggacg tctggggcca agggaccacg gtcaccgtct cctct 345 Evolocumab LCVRsequence (SEQ ID NO:11)cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60tcctgcactg gaaccagcag tgacgttggt ggttataact ctgtctcctg gtaccaacag 120cacccaggca aagcccccaa actcatgatt tatgaggtca gtaatcggcc ctcaggggtt 180tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240caggctgagg acgaggctga ttattactgc aattcatata caagcaccag catggtattc 300ggcggaggga ccaagctgac cgtccta 327

Evolocumab is CAS Registry Number 1256937-27-5.

A variant of evolocumab which does not affect its PCSK9-binding andinhibitory properties is shown in Table 3.

TABLE 3 Evolocumab variant HCVR and LCVR sequences Variant evolocumabHCVR sequence (SEQ ID NO:16)QVQLVQSGAE VKKPGASVKV SCKASGYTLT SYGISWVRQA PGQGLEWMGW VSFYNGNTNY 60AQKLQGRGTM TTDPSTSTAY MELRSLRSDD TAVYYCARGY GMDVWGQGTT VTVSS 115 Variantevolocumab LC sequence (SEQ ID NO:17)QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNSVSWYQQ HPGKAPKLMI YEVSNRPSGV 60SNRFSGSKSG NTASLTISGL QAEDEADYYC NSYTSTSMVF GGGTKLTVL 109

“Pharmaceutical composition” or “pharmaceutical formulation” and thelike means a composition, usually sterile, of a pharmaceutically activedrug, such as a biologically active protein (e.g., a PCSK9-bindingpolypeptide), that is suitable for administration, such as parenteraladministration (including intravenous, intramuscular, subcutaneous,aerosolized, intrapulmonary, intranasal or intrathecal) to a subject inneed thereof and includes only pharmaceutically acceptable excipients,diluents, and other additives deemed safe by the Federal DrugAdministration or other foreign national authorities. Pharmaceuticalformulations include liquid, e.g., aqueous, solutions that can bedirectly administered, and lyophilized powders which can bereconstituted into solutions by adding a diluent before administration.

“Polypeptide” or “protein” means a macromolecule having the amino acidsequence of a native protein, that is, a protein produced by anaturally-occurring and non-recombinant cell; or is produced by agenetically-engineered or recombinant cell, and comprises moleculeshaving the amino acid sequence of the native protein, or moleculeshaving deletions from, additions to, and/or substitutions of one or moreamino acids of the native sequence. The term also embraces amino acidpolymers in which one or more amino acids are chemical analogs of acorresponding naturally-occurring amino acid and polymers. “Polypeptidefragment” refers to a polypeptide that has an amino-terminal deletion, acarboxyl-terminal deletion, and/or an internal deletion as compared withthe full-length native protein. Such fragments can also contain modifiedamino acids as compared with the native protein. Fragments can be aboutfive to 500 amino acids long. For example, fragments can be at least 5,6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450amino acids long. Useful polypeptide fragments include immunologicallyfunctional fragments of antibodies, including binding domains. In thecase of a PCSK9-binding antibody, useful fragments include but are notlimited to a CDR region, a variable domain of a heavy and/or lightchain, a portion of an antibody chain or just its variable regionincluding two CDRs.

To “prevent” (such as the onset of symptoms, a disease, or disorder)does not require the elimination of the possibility of an event. Rather,it denotes that the likelihood of the occurrence of the event has beenreduced in the presence of a compound or method. Preventing in atherapeutic sense includes prophylactic treatments and applications inwhich one reduces the risk that a subject will develop a disorder orother risk factor.

“Stable pharmaceutical formulation,” “stable formulation” or “apharmaceutical formulation is stable” refers to a pharmaceuticalformulation of PCSK9-binding polypeptides that exhibit limited increasedaggregation and/or reduced loss of biological activity of not more than5% when stored at about -30° C. (or colder) to about 5° C. to about 40°C. for at least 1 month, or 2 months, or three months, or 6 months, or 1year, or 2 years, or 5 years, or longer when compared to a controlformulation sample. Formulation stability can be determined by a personof skill in the art using any number of standard assays, includingsize-exclusion HPLC (SEC-HPLC), cation-exchange HPLC (CEX-HPLC),Subvisible Particle Detection by Light Obscuration (“HIAC”) and/orvisual inspection. Typically, the warmer the storage temperature, theshorter the shelf life of the formulation.

Techniques for assessing degradation vary depending upon the identity ofthe protein in the pharmaceutical formulation. Exemplary techniquesinclude size-exclusion chromatography (SEC)-HPLC to detect, e.g.,aggregation, reverse phase (RP)-HPLC to detect, e.g. proteinfragmentation, ion exchange-HPLC to detect, e.g., changes in the chargeof the protein, mass spectrometry, fluorescence spectroscopy, circulardichroism (CD) spectroscopy, Fourier transform infrared spectroscopy(FT-IR), and Raman spectroscopy to detect protein conformationalchanges. All of these techniques can be used singly or in combination toassess the degradation of the protein in the pharmaceutical formulationand determine the shelf life of that formulation. Pharmaceuticalformulations disclosed herein typically exhibit not more than about 2%to about 3% increases in degradation (e.g., fragmentation, aggregationor unfolding) over two years when stored at 2-8° C.

“Subject” or “patient” are used interchangeably and include human andnon-human animal subjects, as well as those with formally diagnoseddisorders, those without formally recognized disorders, those receivingmedical attention, and those at risk of developing disorders.

“Surfactant” means surface-active agents, including substances commonlyreferred to as wetting agents, surface tension depressants, detergents,dispersing agents, emulsifiers, and quaternary ammonium antiseptics.Surfactants are further discussed below.

“Tangential flow filtration,” or “TFF” means a process where a solutionis passed tangentially across an ultrafiltration membrane (i.e., asemi-permeable membrane that can discriminate between molecules ofdifferent size and shape) where lower molecular weight salts and/orsolutes are passed through under pressure.

“Therapeutically effective amount” refers to the amount of a PCSK9antigen binding polypeptide determined to produce a therapeutic responsein a subject. Such therapeutically effective amounts are readilyascertained by one of ordinary skill in the art.

“Trans Membrane Pressure (TMP)” means, in the context of filtration, theaverage differential pressure from the feed to the filtrate side of themembrane and can be expressed by Equation (1):

$\text{TMP}\mspace{6mu} = \mspace{6mu}\frac{\text{FeedPressure+}\mspace{6mu}\text{RetentatePressure}}{2}\mspace{6mu} - \text{PermeatePressure}$

To “treat” and provide “treatment” includes providing therapeutictreatments. Treatment does not require the complete curing of a disorderand encompasses instances in which one reduces symptoms or underlyingrisk factors.

“Ultrafiltration,” “ultrafiltering”, “UF,” and similar terms mean usinga semi-permeable membrane that discriminates between molecules ofdifferent shapes and sizes to separate molecules from differentmolecules, or to concentrate similar, or substantially the same,molecules.

A “variant” of a PCSK9-binding means an amino acid sequence wherein oneor more amino acid residues are inserted into, deleted from and/orsubstituted into the amino acid sequence relative to another polypeptidesequence. Variants include fusion proteins.

“Viscosity” means a fluid’s resistance to flow, and can be measured inunits of centipoise (cP) or milliPascal·second (mPa·s), where 1 cP=1mPa·s, at a given shear rate. Viscosity can be measured by using aviscometer, e.g., Brookfield Engineering (Middleboro, MA) Dial ReadingViscometer, or a rheometer, such as a m-VROC™ rheometer or TAInstruments (New Castle, DE) AESR-G2 cone and plate rheometer. Viscositycan be measured using any other methods and in any other units known inthe art (e.g. absolute, kinematic or dynamic viscosity). Regardless ofthe method used to determine viscosity, the percent reduction inviscosity in excipient formulations versus control formulations willremain approximately the same at a given shear rate.

Components of the Compositions and Methods PCSK9-Binding Polypeptides

The description of PCSK9-binding polypeptides, including evolocumab,have been well described (Chan et al., 2012).

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serineprotease involved in regulating the levels of the low densitylipoprotein receptor (LDLR) protein (Horton, Cohen, & Hobbs, 2007;Seidah & Prat, 2007). PCSK9 is a prohormone-proprotein convertase in thesubtilisin (S8) family of serine proteases (Seidah et al., 2003). Anexemplary human PCSK9 amino acid sequence is shown as SEQ ID NO:3 inTable 1, which is the pre-protein form (unprocessed) of PCSK9. PCSK9proteins can also include fragments of the full length PCSK9 protein.The structure of the PCSK9 protein has been solved (Cunningham et al.,2007; Piper et al., 2007). PCSK9 includes a signal sequence, anN-terminal prodomain, a subtilisin-like catalytic domain, and aC-terminal domain.

PCSK9-binding polypeptides are polypeptides that comprise one or morecomplementary determining regions (CDRs). In some PCSK9-bindingpolypeptides, the CDRs are embedded into a framework region, whichorients the CDR(s) such that the proper PCSK9 binding properties of theCDR(s) is achieved. PCSK9-binding polypeptides can interfere with,block, reduce or modulate the interaction between PCSK9 and LDLR. SuchPCSK9-binding polypeptides are denoted as “neutralizing.” Bindingbetween PCSK9 and LDLR can still occur, even though the PCSK9-bindingpolypeptide is neutralizing and bound to PCSK9. For example, thePCSK9-binding polypeptide prevents or reduces the adverse influence ofPCSK9 on LDLR without blocking the LDLR binding site on PCSK9. Thus, thePCSK9-binding polypeptide can modulate or alter PCSK9’s ability todegrade LDLR, without preventing binding between PCSK9 and LDLR. SuchPCSK9-binding polypeptides can be described as “non-competitivelyneutralizing.” The neutralizing PCSK9-binding polypeptide can bind toPCSK9 in a location or manner that prevents PCSK9 from binding to LDLR.Such PCSK9-binding polypeptides can be described as “competitivelyneutralizing.” PCSK9 neutralizers can result in a greater amount of freeLDLR being present in a subject, resulting in more LDLR binding to LDL,and thereby reducing the amount of LDL in the subject. In turn, thisresults in a reduction in the amount of serum cholesterol present in asubject.

Some PCSK9-binding polypeptides can inhibit PCSK9-mediated activity(including binding). PCSK9-binding polypeptides can also inhibitinteractions between PCSK9 and LDLR and other physiological effectsmediated by PCSK9. PCSK9-binding polypeptides can be human, such asfully human antibodies to PCSK9.

Some PCSK9-binding polypeptides can bind to the catalytic domain ofPCSK9. PCSK9-binding polypeptides can also bind the mature form ofPCSK9. In other cases, PCSK9-binding polypeptides can bind the prodomainof PCSK9. The PCSK9-binding polypeptides, in some cases, can selectivelybind to the mature form of PCSK9. In some cases, PCSK9-binding proteinsbind to the catalytic domain such that PCSK9 cannot bind or bind asefficiently to LDLR. Some PCSK9-binding polypeptides do not bind to theC-terminus of the catalytic domain. In other cases, the PCSK9-bindingpolypeptide does not bind to the N-terminus of the catalytic domain. Inother cases, the PCSK9-binding polypeptide does not bind to the NorC-terminus of the PCSK9 protein. In some cases, the PCSK9-bindingpolypeptide binds to any one of the epitopes bound by anti-PCSK9antibodies. In some cases, this can be determined by competition assaysbetween a candidate antibody and a reference antibody, such asevolocumab. In some cases, the PCSK9-binding polypeptides bind to aspecific conformational state of PCSK9 so as to prevent PCSK9 frominteracting with LDLR. In some cases, the PCSK9-binding polypeptidebinds to the V domain of PCSK9. In some cases, the PCSK9-bindingpolypeptide binds to the V domain of PCSK9 and prevents (or reduces)PCSK9 from binding to LDLR. In some cases, the PCSK9-binding polypeptidebinds to the V domain of PCSK9, and while it does not prevent (orreduce) the binding of PCSK9 to LDLR, the PCSK9-binding polypeptideprevents or reduces the adverse activities mediated through PCSK9 onLDLR.

In some cases, the PCSK9-binding polypeptides comprise one or more CDRs(e.g., 1, 2, 3, 4, 5, or 6 CDRs). In some cases, the PCSK9-bindingpolypeptide comprises (a) a polypeptide structure and (b) one or moreCDRs that are inserted into and/or joined to the polypeptide structure.The polypeptide structure can take a variety of different forms. Forexample, it can be, or comprise, the framework of a naturally occurringantibody, or fragment or variant thereof, or can be synthetic.

The polypeptide structure of PCSK9-binding polypeptides can be anantibody or is derived from an antibody, including monoclonalantibodies, bispecific antibodies, minibodies, domain antibodies,synthetic antibodies (antibody mimetics), chimeric antibodies, humanizedantibodies, antibody fusions (sometimes referred to as “antibodyconjugates”), and portions or fragments of each, respectively. In someinstances, the PCSK9-binding polypeptide is a fragment of an antibody(e.g., a Fab, a Fab′, a F(ab′)₂, or a scFv).

Certain PCSK9-binding polypeptides specifically or selectively bind tohuman PCSK9. In some cases, the PCSK9-binding polypeptide specificallyor selectively binds to human PCSK9 protein having or consisting ofresidues 153-692 of SEQ ID NO:3. In some cases, the PCSK9-bindingpolypeptide specifically binds to at least a fragment of the PCSK9protein and/or a full length PCSK9 protein, with or without a signalsequence.

In some instances, the antibodies include at least one variable heavychain and one variable light chain. In other instances, the antibodiescontain two identical light chains and two identical heavy chains. As anexample, an antibody or PCSK9-binding polypeptide can include a heavychain and a light chain, two heavy chains, or two light chains. In somecases, the PCSK9-binding polypeptide comprises (or consists) of 1, 2,and/or 3 heavy and/or light CDRs from at least one of the sequences (SEQID NOs:4-9) listed in Table 1. In some cases, all six CDRs (CDR1-3 fromthe light (CDRL1, CDRL2, CDRL3) and CDR1-3 from the heavy (CDRH1, CDRH2,and CDRH3)) are part of the PCSK9-binding polypeptide. In some cases, 1,2, 3, 4, 5, or more CDRs are included in the PCSK9-binding polypeptide.In some cases, one heavy and one light CDR from the CDRs in thesequences in Table 1 is included in the PCSK9-binding polypeptide. Insome cases, additional sections are included in the PCSK9-bindingpolypeptide.

The PCSK9-binding polypeptide can be encoded by a nucleic acid sequence,as shown in Table 2 for evolocumab.

In some cases, the PCSK9-binding polypeptide binds to (but does notblock) variants of PCSK9 that are at least 50, 50-60, 60-70, 70-80,80-90, 90-95, 95-99 percent identical, or greater percent identity tothe PCSK9 of SEQ ID NO:3. In some cases, the PCSK9-binding polypeptidebinds to (but does not block) such variants. In some cases, thePCSK9-binding polypeptide binds to and prevents such variants of PCSK9from interacting with LDLR. In some cases, the PCSK9-binding polypeptidebinds to and prevents variants of PCSK9 from interacting with LDLR. Insome cases, the variant of PCSK9 is a human variant, such as variants atposition 474, E620G, and/or E670G. In some cases, the amino acid atposition 474 is valine.

Humanized PCSK9-Binding Polypeptides (e.g., Antibodies) A PCSK9-BindingPolypeptide Can Comprise a Humanized Antibody And/or Part Thereof

A humanized antibody is substantially non-immunogenic in humans and hassubstantially the same affinity for a target as an antibody from anotherspecies from which the humanized antibody is derived.

Antibody modification can be designed to achieve increased bindingaffinity for a target and/or to reduce immunogenicity of the antibody ina recipient. In certain cases, humanized antibodies are modified toeliminate glycosylation sites in order to increase affinity of theantibody for its cognate antigen. Techniques such as “reshaping,”“hyperchimerization,” or “veneering/resurfacing” can be used to producehumanized antibodies. Such techniques typically reduce antibodyimmunogenicity by reducing the number of foreign residues, but do notprevent anti-idiotypic and anti-allotypic responses following repeatedadministration of the antibodies. Other methods of reducingimmunogenicity are known in the art.

The CDRs of the light and heavy chain variable regions of an antibody toPCSK9 can be grafted to framework regions (FRs) from the same, oranother, species. The CDRs of the light and heavy chain variable regionscan be grafted to consensus human FRs. In some cases, the FRs of anantibody to PCSK9 heavy chain or light chain can be replaced with theFRs from a different heavy chain or light chain. The grafted variableregions from an antibody can be used with a constant region that isdifferent from the constant region of an antibody to PCSK9. The graftedvariable regions can be part of a single chain Fv antibody.

PCSK9-Binding Polypeptide Variants

Other antibodies that are useful are variants of the PCSK9-bindingpolypeptides listed above formed by combination or subparts of thevariable heavy and variable light chains shown in Table 1 and comprisevariable light and/or variable heavy chains that each have at least 50%,50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or above99% identity to the amino acid sequences of the sequences in Table 1(either the entire sequence or a subpart of the sequence, e.g., one ormore CDR). In some instances, such antibodies include at least one heavychain and one light chain, whereas in other instances the variant formscontain two identical light chains and two identical heavy chains (orsubparts thereof).

In certain cases, an PCSK9-binding polypeptide comprises a heavy chaincomprising a variable region comprising an amino acid sequence at least90%, at least 95%, or at least 99% identical to an amino acid sequenceof SEQ ID NO:1.

In some cases, the PCSK9-binding polypeptide comprises a sequence thatis at least 90%, 90-95%, and/or 95-99% identical to one or more CDRsfrom the CDRs in at least one of sequences of SEQ ID NOs:4-9. In somecases, 1, 2, 3, 4, 5, or 6 CDRs (each being at least 90%, 90-95%, and/or95-99% identical to the above sequences) is present.

In certain cases, an PCSK9-binding polypeptide comprises a light chaincomprising a variable region comprising an amino acid sequence at least90%, at least 95%, or at least 99% identical to an amino acid sequenceof SEQ ID NO:11 or 15.

In other cases, an PCSK9-binding polypeptide comprises a heavy chaincomprising a variable region comprising an amino acid sequence at least90%, at least 95%, or at least 99% identical to an amino acid sequenceof SEQ ID NO:10 or 14.

A skilled artisan is able to determine suitable variants ofPCSK9-binding polypeptides using well-known techniques. In certaincases, one skilled in the art can identify suitable areas of themolecule that can be changed without destroying activity by targetingregions not believed to be important for activity. In certain cases, onecan identify residues and portions of the molecules that are conservedamong similar polypeptides. In certain cases, even areas that can beimportant for biological activity or for structure can be subject toconservative amino acid substitutions without destroying the biologicalactivity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues which are important for activity or structure insimilar proteins. One skilled in the art can opt for chemically similaramino acid substitutions for such predicted important amino acidresidues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarPCSK9-binding polypeptides. In view of such information, one skilled inthe art can predict the alignment of amino acid residues of an antibodywith respect to its three-dimensional structure. In certain cases, oneskilled in the art can choose not to make radical changes to amino acidresidues predicted to be on the surface of the protein, since suchresidues can be involved in important interactions with other molecules.Moreover, one skilled in the art can generate test variants containing asingle amino acid substitution at each desired amino acid residue. Thevariants can then be screened using activity assays known in the art.Such variants can be used to gather information about suitable variants.For example, if one observed that a change to a particular amino acidresidue resulted in destroyed, undesirably reduced, or unsuitableactivity, variants with such a change can be avoided. In other words,based on information gathered from such routine experiments, one skilledin the art can readily determine the amino acids where furthersubstitutions should be avoided either alone or in combination withother mutations.

In certain cases, PCSK9-binding polypeptide variants includeglycosylation variants wherein the number and/or type of glycosylationsite has been altered compared to the amino acid sequences of a parentpolypeptide. In certain cases, protein variants comprise a greater or alesser number of N-linked glycosylation sites than the native protein.Additional preferred antibody variants include cysteine variants whereinone or more cysteine residues are deleted from or substituted foranother amino acid (e.g., serine) as compared to the parent amino acidsequence. Cysteine variants can be useful when antibodies must berefolded into a biologically active conformation such as after theisolation of insoluble inclusion bodies. Cysteine variants generallyhave fewer cysteine residues than the native protein, and typically havean even number to minimize interactions resulting from unpairedcysteines.

Competing PCSK9-Binding Polypeptides

PCSK9-binding polypeptides that compete with evolocumab or functionalfragments binding to an epitope bound by evolocumab for specific bindingto PCSK9 can be used. Such PCSK9-binding polypeptides can also bind tothe same epitope PCSK9-binding polypeptides or an overlapping epitope.PCSK9-binding polypeptides and fragments that compete with or bind tothe same epitope as evolocumab show similar functional properties. Thus,as a specific example, the PCSK9-binding polypeptides that are providedinclude those that compete with an antibody or PCSK9-binding polypeptidehaving all six of the CDRs of evolocumab (SEQ ID NOs:4-9) or two lightchains and two heavy chains of SEQ ID NOs:2 and 1, respectively).

Exemplary Epitopes

Epitopes of SEQ ID NO:3 (human PCSK9 polypeptide) to which anti-PCSK9antibodies bind are provided. In the case of evolocumab (and theevolocumab variant, having HCVR of SEQ ID NO:14 and a LCVR of SEQ IDNO:15), they are S153, D188, I189, Q190, S191, D192, R194, E197, G198,R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379,I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239,G244, M247, I369, S372, C375, C378, R237, and D238.

Preparation of PCSK9-Binding Polypeptides (e.g., Antibodies)

Certain strategies can be used to manipulate the inherent properties ofan antibody, such as the affinity of an antibody for its target. Suchstrategies include the use of site-specific or random mutagenesis of thepolynucleotide molecule encoding an antibody to generate an antibodyvariant. In certain cases, such generation is followed by screening forantibody variants that exhibit the desired change, e.g. increased ordecreased affinity.

The amino acid residues targeted in mutagenic strategies can be those inthe CDRs or the FRs.

In certain cases, smaller and more effectively screened libraries ofantibody variants are produced by restricting random or site-directedmutagenesis to hyper-mutation sites in the CDRs, which are sites thatcorrespond to areas prone to mutation during the somatic affinitymaturation process.

Antibodies can be expressed in cell lines. Sequences (such aspolynucleotides encoding the polypeptides of SEQ ID NOs:1 and 2, such asthe polynucleotides of SEQ ID NOs:12 and 13) encoding particularantibodies can be used to transform a suitable mammalian host cell.Transformation can be by any known method for introducingpolynucleotides into a host cell. The transformation procedure useddepends upon the host to be transformed. Methods for introducingheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), humanepithelial kidney 293 cells, and a number of other cell lines.

In certain cases, antibodies are produced by the 21B12 hybridoma cellline (Jackson et al., 2009). In certain cases, PCSK9-bindingpolypeptides bind to PCSK9 with a dissociation constant (K_(D)) of lessthan approximately 1 nM, e.g., 1000 pM to 100 pM, 100 pM to 10 pM, 10 pMto 1 pM, and/or 1 pM to 0.1 pM or less.

PCSK9-binding polypeptides can comprise an immunoglobulin molecule of atleast one of the IgG1, IgG2, IgG3, IgG4, Ig E, IgA, IgD, and IgMisotype. In certain cases, PCSK9-binding polypeptides comprise a humankappa light chain and/or a human heavy chain. In certain cases, theheavy chain is of the IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, or IgMisotype. In certain cases, PCSK9-binding polypeptides have been clonedfor expression in mammalian cells. In certain cases, PCSK9-bindingpolypeptides comprise a constant region other than any of the constantregions of the IgG1, IgG2, IgG3, IgG4, IgE, IgA, IgD, and IgM isotype.

In certain cases, PCSK9-binding polypeptides comprise a human lambdalight chain and a human IgG2 heavy chain. In certain cases,PCSK9-binding polypeptides comprise a human lambda light chain and ahuman IgG4 heavy chain. In certain cases, PCSK9-binding polypeptidescomprise a human lambda light chain and a human IgG1, IgG3, IgE, IgA,IgD or IgM heavy chain. In other embodiments, PCSK9-binding polypeptidescomprise a human kappa light chain and a human IgG2 heavy chain. Incertain cases, PCSK9-binding polypeptides comprise a human kappa lightchain and a human IgG4 heavy chain. In certain cases, PCSK9-bindingpolypeptides comprise a human kappa light chain and a human IgG1, IgG3,IgE, IgA, IgD or IgM heavy chain. In certain cases, PCSK9-bindingpolypeptides comprise variable regions of antibodies ligated to aconstant region that is neither the constant region for the IgG2isotype, nor the constant region for the lgG4 isotype. In certain cases,PCSK9-binding polypeptides have been cloned for expression in mammaliancells.

In the case of evolocumab, the antibody is an IgG2-lambda humanmonoclonal antibody; gamma 2 heavy chain -disulfide with lambda lightchain tetrakisdisulfide. Evolocumab is glycosylated at Asn-291 andAsn-291″ and has disulfide bridges between residues 22′-90′, 22″-90″,22′-96′, 22″-96″, 129-214′, 129″-214″, 137′-196′, 137″-196″, 142-198,142″-198″, 217-217′, 218-218′, 221-221″, 224-224″, 255-315, 255″-315″,361-419, and 361″-419″.

In certain cases, conservative modifications to the heavy and lightchains of evolocumab or to those of an antibody having HCVR and LCVR ofSEQ ID NOs:14 and 15 can produce antibodies to PCSK9 having functionaland chemical characteristics similar to those of the antibodies from thehybridoma line 21B12. In contrast, substantial modifications in thefunctional or chemical characteristics of antibodies to PCSK9 can beaccomplished by selecting substitutions in the amino acid sequence ofthe heavy and light chains that differ significantly in their effect onmaintaining (a) the structure of the molecular backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain.

For example, a conservative amino acid substitution can involve asubstitution of a native amino acid residue with a nonnative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position.

PCSK9-binding polypeptides often comprise one or more polypeptides. Anyof a variety of expression vector/host systems can be used to expresspolynucleotide molecules encoding polypeptides comprising one or morePCSK9-binding polypeptide components or the PCSK9-binding polypeptideitself. Such systems include microorganisms, such as bacteriatransformed with recombinant bacteriophage, plasmid, or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transfected with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus,TMV) or transformed with bacterial expression vectors (e.g., Ti orpBR322 plasmid); or animal cell systems.

A polypeptide comprising one or more PCSK9-binding polypeptidecomponents or the PCSK9-binding polypeptide itself can be purified fromthe various expression systems; such techniques are well-known to thoseof skill in the art.

Pharmaceutical Formulation Components

In some embodiments, the pharmaceutical formulation comprising aPCSK9-binding polypeptide comprises more than one differentPCSK9-binding polypeptide. In certain embodiments, pharmaceuticalformulations comprise more than one PCSK9-binding polypeptide whereinthe antigen binding proteins to PCSK9 bind more than one epitope. Insome embodiments, the various antigen binding proteins will not competewith one another for binding to PCSK9. In some embodiments, thepharmaceutical formulation comprises evolocumab.

A PCSK9-binding polypeptide can be linked to a half-life extendingvehicle. Such vehicles include polyethylene glycol (PEG), glycogen(e.g., glycosylation of the PCSK9-binding polypeptide), and dextran.

Acceptable formulation components preferably are nontoxic to recipientsat the dosages and concentrations used. Pharmaceutical formulations cancomprise agents for modifying, maintaining or preserving, for example,the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,sterility, stability, rate of dissolution or release, adsorption orpenetration of the composition.

For example, suitable formulation materials include amino acids (such asproline, arginine, lysine, methionine, taurine, glycine, glutamine, orasparagine); antimicrobials; antioxidants (such as ascorbic acid, sodiumsulfite or sodium hydrogen-sulfite); buffers (such as borate,bicarbonate, sodium phosphate, sodium acetate (“NaOAC”), Tris-HCl, Trisbuffer, citrates, phosphate buffer, phosphate-buffered saline (i.e., PBSbuffer) or other organic acids); bulking agents (such as mannitol orglycine); chelating agents (such as ethylenediamine tetra acetic acid(EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone,beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;monosaccharides; disaccharides; and other carbohydrates (such asglucose, sucrose,, fructose, lactose, mannose, trehelose, or dextrins);proteins (such as serum albumin, gelatin or immunoglobulins); coloring,flavoring and diluting agents; emulsifying agents; hydrophilic polymers(such as polyvinylpyrrolidone); low molecular weight polypeptides;salt-forming counter ions (such as sodium); preservatives (such asbenzalkonium chloride, benzoic acid, salicylic acid, thimerosal,phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbicacid or hydrogen peroxide); solvents (such as glycerin, propylene glycolor polyethylene glycol); sugar alcohols (such as mannitol or sorbitol);suspending agents; surfactants or wetting agents (such as pluronics,PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stabilityenhancing agents (such as sucrose or sorbitol); tonicity enhancingagents (such as alkali metal halides, preferably sodium or potassiumchloride, mannitol sorbitol); delivery vehicles; diluents; excipientsand/or pharmaceutical adjuvants.

In one aspect, the pharmaceutical formulation comprises highconcentrations of PCSK9-binding polypeptide. In certain embodiments, thePCSK9-binding polypeptide concentration ranges from about 70 mg/mL toabout 260 mg/mL, e.g., about 70 mg/mL, about 80 mg/mL, about 90 mg/mL,about 100 mg/mL, about 110 mg/mL, about 120 mg/mL, about 130 mg/mL,about 140 mg/mL, about 150 mg/mL, about 160 mg/mL, about 170 mg/mL,about 180 mg/mL, about 190 mg/mL, about 200 mg/mL, about 210 mg/mL,about 220 mg/mL, about 230 mg/mL, about 240 mg/mL, about 250 mg/mL, orabout 260 mg/mL. In some embodiments, the concentration of evolocumabranges from about 140 mg/mL to about 210 mg/mL, e.g., about 140 mg/mL,about 150 mg/mL, about 160 mg/mL, about 170 mg/mL, about 180 mg/mL,about 190 mg/mL, about 200 mg/mL, about 210 mg/mL, about 220 mg/mL,about 230 mg/mL, about 240 mg/mL, about 250 mg/mL, or about 260 mg/mL.

In another aspect, the pharmaceutical formulation comprises at least onebuffering agent such as, for example, sodium acetate, phosphates,phosphate buffered saline (“PBS”), histidine, and/or Tris buffer ofabout pH 7.0-8.5. The buffer serves to maintain a physiologicallysuitable pH. In addition, the buffer can enhance isotonicity andchemical stability of the pharmaceutical formulation. In certainembodiments, the buffering agent ranges from about 5 mM to about 100 mM,e.g., about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 30 mM,about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about90 mM, or about 100 nM buffering agent. In certain embodiments, thebuffering agent is NaOAC. In certain embodiments, the buffering agent isNaOAC and is present at a concentration of about 10 mM. In otherembodiments, the buffer is sodium glutamate. In certain embodiments, thebuffering agent is sodium glutamate and is present at a concentration ofabout 10 mM. In yet other embodiments, the buffering agent is aphosphate buffer. In certain embodiments, the phosphate buffer ispresent at a concentration of about 10 mM. In yet further embodiments,the buffering agent is histidine. In some of these embodiments, thehistidine buffer is present at a concentration of about 10 mM. Useful pHvalues of the pharmaceutical formulation include from about 4 to about7, or from about 4.8 to about 6.9, or from about 5.0 to about 5.5, orabout 5, or about 5.4.

In certain embodiments, the pharmaceutical formulation is isotonic withan osmolality ranging from between about 250 to about 400 mOsm/kg, e.g.,about 250 mOsm/kg, about 260 mOsm/kg, about 270 mOsm/kg, about 280mOsm/kg, about 290 mOsm/kg, about 300 mOsm/kg, about 310 mOsm/kg, about320 mOsm/kg, about 330 mOsm/kg, about 340 mOsm/kg, about 350 mOsm/kg,about 360 mOsm/kg, about 370 mOsm/kg, about 380 mOsm/kg, about 390mOsm/kg, or about 400 mOsm/kg. Osmolality is the measure of the ratio ofsolutes to volume fluid. In other words, it is the number of moleculesand ions (or molecules) per kilogram of a solution. In certainembodiments, the osmolality is 300 mOsm/kg. Osmolality may be measuredby an osmometer, such as Advanced Instruments 2020 Multi-sampleOsmometer, Norwood, MA. The Advanced Instruments 2020 Multi-sampleOsmometer measures osmolality by using the Freezing Point Depressionmethod. The higher the osmolytes in a solution, the temperature in whichit will freeze drops. Osmolality may also be measured using any othermethods and in any other units known in the art such as linearextrapolation. In other embodiments, the pharmaceutical formulation isisotonic to a human blood cell, such as a red blood cell.

In still another aspect, the pharmaceutical formulation comprises atleast one surfactant polyoxyethylenesorbitan monooleate (polysorbate 80or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer(Poloxamers such as Pluronic® F-68 and other Pluronics®), Sorbitan alkylesters (Spans®) Polyethylene glycol octylphenyl ethers (Triton X-100),Polyethylene glycol alkyl ethers (Brij), Polypropylene glycol alkylethers, Glucoside alkyl ethers, and D-α-tocopherol polyethylene glycolsuccinate (vitamin E TPGS). In certain embodiments, the pharmaceuticalformulation comprises a surfactant at a concentration that ranges fromabout 0.0001% to about 10% weight per volume (w/v) of the formulation,e.g., about 0.0001%, about 0.005%, about 0.006%, about 0.007%, about0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.5%,about 1%, about 5%, or about 10% surfactant (w/v) of the formulation. Incertain embodiments, the pharmaceutical formulation comprisespolysorbate 80 at a concentration that ranges from about 0.0001% toabout 1% w/v of the formulation. In certain embodiments, thepharmaceutical formulation comprises polysorbate 80 at a concentrationat about 0.01% w/v of the formulation. In other embodiments, theformulation comprises Pluronic® F-68 at a concentration that ranges fromabout 0.0001% to about 1% w/v of the formulation. In certainembodiments, the pharmaceutical formulation comprises Pluronic® F-68 ata concentration at about 0.01% w/v of the formulation. In still otherembodiments, the formulation comprises vitamin E TPGS at a concentrationthat ranges from about 0.0001% to about 1% w/v of the formulation. Incertain embodiments, the pharmaceutical formulation comprises vitamin ETPGS at a concentration at about 0.01% w/v of the formulation.

The pharmaceutical formulation can comprise at least one stabilizingagent, such as a polyhydroxy hydrocarbon (including sorbitol, mannitol,glycerol and dulcitol) and/or a disaccharide (including sucrose,lactose, maltose and trehalose) and/or an amino acid (beyond thoseformulations including an arginine salt, such as argininemonohydrochloride, and an acetyl derivative of arginine, such asN-acetyl arginine and can include, for example, proline, lysine,methionine, and taurine) and or benzyl alcohol; the total of saidpolyhydroxy hydrocarbon and/or disaccharide and/or amino acid and/orbenzyl alcohol being about 0.5% to about 10% w/v of the formulation. Thepharmaceutical formulation can comprise proline, for example, at about10 mM to about 200 mM, such as from about 50 mM to about 150 mM, such asfrom about 90 mM to about 120 mM, such as about 120 mM.

In one aspect, the pharmaceutical formulation has a viscosity level ofless than about 80 centipoise (cP) as measured at room temperature(i.e., 25° C.). In certain embodiments, the pharmaceutical formulationhas a viscosity level of less than about 70 cP, about 60 cP, about 50cP, about 40 cP, about 30 cP, about 25 cP, about 20 cP, about 18 cP,about 15 cP, about 12 cP, about 10 cP; about 8 cP, about 6 cP, about 4cP; about 2 cP; or about 1 cP.

In one aspect, the pharmaceutical formulation is stable as measured byat least one stability assay, such as an assay that examines thebiophysical or biochemical characteristics of the PCSK9-bindingpolypeptide over time. Pharmaceutical formulation stability can bemeasured using SEC-HPLC. SEC-HPLC separates proteins based ondifferences in their hydrodynamic volumes. Molecules with largerhydrodynamic proteins volumes elute earlier than molecules with smallervolumes. In the case of SEC-HPLC, a stable pharmaceutical formulationexhibits no more than about a 5% increase in HMW species as compared toa control sample, such as, for example no more than about a 4%, no morethan about a 3%, no more than about a 2%, no more than about a 1%, nomore than about a 0.5% increase in HMW species as compared to a controlsample.

Alternatively, or in addition, stability can be measured usingcation-exchange HPLC (CEX-HPLC). CEX-HPLC separates proteins based ondifferences in their surface charge. At a set pH, charged isoforms of ananti-PCSK9 ABP are separated on a cation-exchange column and elutedusing a salt gradient. The eluent is monitored by ultraviolet light (UV)absorbance. The charged isoform distribution is evaluated by determiningthe peak area of each isoform as a percent of the total peak area. Inthe case of CEX-HPLC, a stable pharmaceutical formulation exhibits nomore than about a 5% decrease in the main isoform peak as compared to acontrol sample, such as, for example, no more than about a 3% to about a5% decrease in the main isoform peak as compared to a control sample; nomore than about a 4% decrease, no more than about a 3% decrease, no morethan about a 2% decrease, no more than about a 1% decrease, no more thanabout a 0.5% decrease in the main isoform peak as compared to a controlsample.

Also alternatively, or in addition, formulation stability can bemeasured using Subvisible Particle Detection by Light Obscuration(HIAC). An electronic, liquid-borne particle-counting system (HIAC/Royco9703 (Hach Company; Loveland, CO) or equivalent) containing alight-obscuration sensor (HIAC/Royco HRLD-150 or equivalent) with aliquid sampler quantifies the number of particles and their size rangein a given test sample. When particles in a liquid pass between thelight source and the detector they diminish or “obscure” the beam oflight that falls on the detector. When the concentration of particleslies within the normal range of the sensor, these particles are detectedone-by-one. The passage of each particle through the detection zonereduces the incident light on the photo-detector and the voltage outputof the photo-detector is momentarily reduced. The changes in the voltageregister as electrical pulses that are converted by the instrument intothe number of particles present. The method is nonspecific and measuresparticles regardless of their origin. Particle sizes monitored aregenerally 10 µm, and 25 µm. In the case of HIAC, a stable pharmaceuticalformulation exhibits no more than 6000 10 µm particles per container (orunit), as compared to a control sample, such as, for example no morethan 5000, no more than 4000, no more than 3000, no more than 2000, nomore than 1000, 10 µm particles per container (or unit) as compared to acontrol sample. In other cases, a stable pharmaceutical formulationexhibits no more than 600 25 µm particles per container (or unit) ascompared to a control sample, such as, for example, no more than 500, nomore than 400, no more than 300, no more than 200, no more than 100, nomore than 50 25 µm particles per container (or unit) as compared to acontrol sample.

Pharmaceutical formulation stability can also be assessed using visualassessment. Visual assessment is a qualitative method used to describethe visible physical characteristics of a sample. The sample is viewedagainst a black and/or white background of an inspection booth,depending on the characteristic being evaluated (e.g., color, clarity,presence of particles or foreign matter). Samples are also viewedagainst an opalescent reference standard and color reference standards.In the case of visual assessment, a stable pharmaceutical formulationexhibits no significant change in color, clarity, presence of particlesor foreign matter as compared to a control sample.

Exemplary Pharmaceutical Formulations

Shown in Table 4 are exemplary pharmaceutical formulations ofPCSK9-binding polypeptides. In some of the formulations, ranges aregiven, and in the sub-examples (e.g., 1.1), a specific example is given.

TABLE 4 Exemplary pharmaceutical formulations of PCSK9-bindingpolypeptides Example PCSK9-binding polypeptide Buffer ExcipientsSurfactant Final pH Viscosity @1000/s (cP)) Osmolality 1 195-227 mg/mL10 mM NaOAc 140 mM NAR 63 mM Arg HCl 0.01% (w/v) polysorbate 80 5.2 ~80~270 mOsm/kg 1.1 227 mg/mL 10 mM NaOAc 140 mM NAR 63 mM Arg HCl 0.01%(w/v) polysorbate 80 5.2 77.4 269 2 195-227 mg/mL 10 mM NaOAc 155 mM NAR70 mM Arg HCl 0.01% (w/v) polysorbate 80 5.4 ~50 ~300 2.1 218 mg/mL 10mM NaOAc 170 mM NAR 63 mM Arg HC 0.01% (w/v) polysorbate 80 5.6 49.6 cP302 3 195-227 mg/mL 10 mM NaOAc 170 mM NAR 63 mM Arg HCl 0.01% (w/v)polysorbate 80 5.6 ~50 cP ~300 mOsm/kg 3.1 222 mg/mL 10 mM NaOAc 170 mMNAR 63 mM Arg HCl 0.01% (w/v) polysorbate 80 5.6 52.3 cP 296 mOsm/kg 4195-227 mg/mL 10 mM NaOAc 140 mM NAR 50 mM Arg HC 0.005%-0.015%polysorbate 80 5.1-5.7 4.1 210 mg/mL 10 mM NaOAc 140 mM NAR 50 mM ArgHCl 0.01% (w/v) polysorbate 80 5.4 ~40 cP 5 188-190 mg/mL 10 mM NaOAc155 mM NAR 120 mM proline 0.01% (w/v) polysorbate 80 5.4 290 ~23 5.1 190mg/mL 10 mM NaOAc 155 mM NAR 120 mM proline 0.01% (w/v) polysorbate 805.4 290 22.8 6 200-201 mg/mL 10 mM NaOAc 155 mM NAR 120 mM proline 0.01%(w/v) polysorbate 80 5.4 295 ~35 6.1 200 mg/mL 10 mM NaOAc 155 mM NAR120 mM proline 0.01% (w/v) polysorbate 80 5.4 295 34.5 7 210-214 mg/mL10 mM NaOAc 155 mM NAR 120 mM proline 0.01% (w/v) polysorbate 80 5.4 298~50 7.1 210 mg/mL 10 mM NaOAc 155 mM NAR 120 mM proline 0.01% (w/v)polysorbate 80 5.4 298 51.4 *expected variability in concentrationmeasurements, formulation process and viscosity measurements wereobserved

Therapeutic Applications

PCSK9-binding polypeptides such as evolocumab can be used in a varietyof therapeutic applications. For example, PCSK9-binding polypeptides areuseful for treating conditions associated with PCSK9, such ascholesterol related disorders (serum cholesterol related disorders) suchas hypercholesterolemia. PCSK9-binding polypeptides can be useful intreating consequences, symptoms, and/or the pathology associated withPCSK9 activity.

Disorders that relate to, involve, or can be influenced by elevatedlevels of molecules, or groups of molecules, including cholesterol(including serum cholesterol), LDL, LDLR, PCSK9, VLDL-C, apoprotein B(“ApoB”), lipoprotein A (“Lp(a)”), triglycerides, HDL-C, non-HDL-C, andtotal cholesterol levels can be addressed by methods that use theevolocumab pharmaceutical compositions disclosed herein to treat and/orprevent and/or reduce the risk of such disorders in a subject. Thedisclosed evolocumab compositions can be used to modulate the levels ofthese molecules or groups of molecules, such as reducing the amount ofthese molecules or groups of molecules. For example, the disclosedevolocumab compositions can be used in methods to decrease the amount ofthese molecules or groups of these molecules from an abnormally highlevel or from even a normal level, that is, the amount of cholesterol(including serum cholesterol), LDL, LDLR, PCSK9, VLDL-C, ApoB, Lp(a),triglycerides, HDL-C, non-HDL-C, and total cholesterol levels can bereduced.

A “cholesterol related disorder” (which includes “serum cholesterolrelated disorders”) includes any one or more of the following: familialhypercholesterolemia, non-familial hypercholesterolemia, hyperlipidemia,heart disease, metabolic syndrome, diabetes, coronary heart disease,stroke, cardiovascular diseases, Alzheimer’s disease and generallydyslipidemias, which can be manifested, for example, by an elevatedtotal serum cholesterol, elevated LDL, elevated triglycerides, elevatedVLDL, and/or low HDL. Some non-limiting examples of primary andsecondary dyslipidemias include metabolic syndrome, diabetes mellitus,familial combined hyperlipidemia, familial hypertriglyceridemia,familial hypercholesterolemias, including heterozygoushypercholesterolemia, homozygous hypercholesterolemia, familialdefective apop lipoprotein B-100; polygenic hypercholesterolemia;remnant removal disease, hepatic lipase deficiency; dyslipidemiasecondary to any of the following: dietary indiscretion, hypothyroidism,drugs including estrogen and progestin therapy, beta-blockers, andthiazide diuretics; nephrotic syndrome, chronic renal failure, Cushing’ssyndrome, primary biliary cirrhosis, glycogen storage diseases,hepatoma, cholestasis, acromegaly, insulinoma, isolated growth hormonedeficiency, and alcohol-induced hypertriglyceridemia. The disclosedevolocumab compositions can also be used to treat and/or prevent and/orreduce the risk of atherosclerotic diseases, such as cardiovasculardeath, non-cardiovascular or all-cause death, coronary heart disease,coronary artery disease, peripheral arterial disease, stroke (ischemicand hemorrhagic), angina pectoris, or cerebrovascular disease and acutecoronary syndrome, myocardial infarction and unstable angina. Thedisclosed evolocumab compositions can also be useful in reducing therisk of fatal and nonfatal heart attacks, fatal and non-fatal strokes,certain types of heart surgery, hospitalization for heart failure, chestpain in patients with heart disease, and/or cardiovascular eventsbecause of established heart disease such as prior heart attack, priorheart surgery, and/or chest pain with evidence of clogged arteriesand/or transplant-related vascular disease. In some cases, the disclosedevolocumab compositions can be used in methods of preventing or reducingthe cardiovascular risk due to elevated CRP or hsCRP. In someembodiments, the ABP and methods can be used to reduce the risk ofrecurrent cardiovascular events.

Diseases or disorders that are generally addressable (either treatableor preventable) through the use of statins can also benefit from theapplication of the disclosed evolocumab compositions. Futhermore,disorders or diseases that can benefit from the prevention ofcholesterol synthesis or increased LDLR expression can also be treatedusing the disclosed evolocumab compositions. In addition, the use of thedisclosed evolocumab compositions can be especially useful in thetreatment of diabetes. Not only is diabetes a risk factor for coronaryheart disease, but insulin increases the expression of PCSK9. That is,people with diabetes have elevated plasma lipid levels (which can berelated to high PCSK9 levels) and can benefit from lowering thoselevels.

Where a PCSK9-binding polypeptide is used for therapeutic applications,a PCSK9-binding polypeptide can inhibit, interfere with, or modulate oneor more biological activities of PCSK9. For example, a PCSK9-bindingpolypeptide can bind specifically to human PCSK9 and/or substantiallyinhibit binding of human PCSK9 to LDLR by at least about 20%-40%,40-60%, 60-80%, 80-85%, or more (for example, by measuring binding in anin vitro competitive binding assay). In some cases, the PCSK9-bindingpolypeptide has a K_(d) of less (binding more tightly) than 10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ M. In some cases, the PCSK9-bindingpolypeptide has an IC50 for blocking the binding of LDLR to PCSK9 ofless than 1 µM, 1000 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1000pM to 500 pM, 500 pM to 200 pM, less than 200 pM, 200 pM to 150 pM, 200pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM.

Pharmaceutical formulations can be administered in combination therapy,i.e., combined with other agents. The combination therapy can comprise aPCSK9-binding polypeptide in combination with at least oneanti-cholesterol agent. Agents include in vitro synthetically preparedchemical formulations, antibodies, antigen binding regions, andcombinations and conjugates thereof. In certain embodiments, an agentcan act as an agonist, antagonist, allosteric modulator, or toxin. Incertain embodiments, an agent can act to inhibit or stimulate its target(e.g., receptor or enzyme activation or inhibition), and thereby promoteincreased expression of LDLR or decrease serum cholesterol levels.

A PCSK9-binding polypeptide can be administered prior to, concurrentwith, and subsequent to treatment with a cholesterol-lowering (serumand/or total cholesterol) agent. For example, a PCSK9-bindingpolypeptide can be administered prophylactically to prevent or mitigatethe onset of hypercholesterolemia, heart disease, diabetes, and/or anycholesterol related disorder. Furthermore, a PCSK9-binding polypeptidecan be administered for the treatment of an existinghypercholesterolemia condition. In some cases, administration of aPCSK9-binding polypeptide can delay the onset of the disorder and/orsymptoms associated with the disorder. In some cases, the PCSK9-bindingpolypeptide is provided to a subject lacking any symptoms of any one ofthe cholesterol related disorders or a subset thereof.

A PCSK9-binding polypeptide can be used with particular therapeuticagents to treat various cholesterol related disorders, such ashypercholesterolemia. In view of the condition and the desired level oftreatment, two, three, or more agents can be administered. Such agentscan be provided together by inclusion in the same formulation.Alternatively, such agents can be formulated separately and, if desired,provided together by inclusion in a treatment kit. In another example,such agents can be provided separately.

Dosage and Dosing Regimens

The amount of a PCSK9-binding polypeptide, such as a mAb, such asevolocumab, administered to a patient is a therapeutically effectiveamount. A typical dosage of a PCSK9-binding protein can range from about0.1 µg/kg to up to about 100 mg/kg or more. In certain cases, the dosagecan range from 0.1 µg/kg up to about 100 mg/kg; or 1 µg/kg up to about100 mg/kg; or 5 µg/kg up to about 100 mg/kg; or 1 mg/kg to about 50mg/kg; or 2 mg/kg to about 20 mg/kg; or 2 mg/kg to about 10 mg/kg ofPCSK9-binding polypeptide.

The amount (or dose) of PCSK9-binding polypeptide can range from atleast about 10 mg to at about 1400 mg; or about 14 mg to about 1200 mg;or about 14 mg to about 1000 mg; or about 14 mg to about 800 mg; orabout 14 mg to about 700 mg; or about 14 mg to about 480 mg; or about 20mg up to about 480 mg; or about 70 mg up to about 480 mg; or about 80 mgto about 480 mg; or about 90 mg to about 480 mg; or about 100 mg toabout 480 mg, or about 105 mg to about 480 mg; or about 110 mg to about480 mg; or about 115 mg to about 480 mg; or about 120 mg to about 480mg; or about 125 mg to about 480 mg; or about 130 mg to about 480 mg; orabout 135 mg to about 480 mg; or about 140 mg to about 480 mg; or about145 mg to about 480 mg; or about 150 mg to about 480 mg; or about 160 mgto about 480 mg; or about 170 mg to about 480 mg; or about 180 mg toabout 480 mg or about 190 mg to about 480 mg or about 200 mg to about480 mg; or about 210 mg to about 480 mg; or about 220 mg to about 480mg; or about 230 mg to about 480 mg; or about 240 mg to about 480 mg; orabout 250 mg to about 480 mg; or about 260 mg to about 480 mg; or about270 mg to about 480 mg; or about 280 mg to about 480 mg; or about 290 mgto about 480 mg; or about 300 mg to about 480 mg; or about 310 mg toabout 480 mg; or about 320 mg to about 480 mg; or about 330 mg to about480 mg; or about 340 mg to about 480 mg; or about 350 mg to about 480mg; or about 360 mg to about 480 mg; or about 370 mg to about 480 mg; orabout 380 mg to about 480 mg; or about 390 mg to about 480 mg; or about400 mg to about 480 mg; or about 410 mg to about 480 mg; or about 420 mgto about 480 mg; or about 430 mg to about 480 mg; or about 440 mg toabout 480 mg; or about 450 mg to about 480 mg; or about 460 mg to about480 mg; or about 470 mg to about 480 mg of PCSK9-binding polypeptide.

The frequency of dosing will take into account the pharmacokineticparameters of a PCSK9-binding polypeptide and/or any additionaltherapeutic agents in the formulation. A clinician can administer theformulation until a dosage is reached that achieves the desired effect.The formulation can be administered as a single dose, or as two, three,four or more doses (which may or may not contain the same amount of thePCSK9-binding polypeptide) over time, or as a continuous infusion via animplantation device or catheter. The formulation can also be deliveredsubcutaneously or intravenously with a needle and syringe. With respectto subcutaneous delivery, pen delivery devices, as well as body injectorand autoinjector delivery devices, can deliver pharmaceuticalformulations comprising PCSK9-binding polypeptides.

In certain cases, a dose of at least about 10 mg; or up to about 14 mg;or up to about 20 mg; or up to about 35 mg; or up to about 40 mg, or upto about 45 mg, or up to about 50 mg; or up to about 70 mg of anPCSK9-binding polypeptide is administered once a week (QW) to a patientin need thereof.

In other cases, a dose of at least about 70 mg, or up to about 100 mg;or up to about 105 mg, or up to about 110 mg; or up to about 115 mg, orup to about 120 mg; or up to about 140 mg; or up to about 160 mg; or upto about 200 mg; or up to about 250 mg; or up to 280 mg; or up to 300mg; or up to 350 mg; or up to 400 mg; or up to 420 mg of anPCSK9-binding polypeptide is administered once every other week, (orevery two weeks; “Q2W:”), to a patient in need thereof.

In certain other cases, a dose of at least about 250 mg; or up to about280 mg; or up to about 300 mg; or up to about 350 mg; or up to about 400mg; or up to about 420 mg; or up to about 450 mg; or up to 480 mg of aan PCSK9-binding polypeptide is administered once every four weeks(“Q4W”), (or once a calendar month) to a patient in need thereof.

For example, evolocumab can be administered Q2W as 210 mg doses.Alternatively, evolocumab can be administered Q4W as 420 mg doses.Depending on the circumstances of the condition being treated, boththese drug doses can be administered weekly.

In some cases, the serum LDL cholesterol level is reduced by at leastabout 15%, as compared to a pre-dose serum LDL cholesterol level. Insome embodiments, the serum LDL cholesterol level is reduced by at leastabout 20%, or by at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or even more.

Storage and Kits

Formulations comprising a PCSK9-binding polypeptide, with or without atleast one additional therapeutic agents, can be prepared for storage bymixing the selected formulation having the desired degree of purity withoptional formulation agents in the form of a lyophilized cake or anaqueous solution. Further, a formulation comprising a PCSK9-bindingpolypeptide, with or without at least one additional therapeutic agent,can be formulated as a lyophilisate using appropriate excipients.

Once the pharmaceutical formulation has been formulated, it can bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations can bestored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. In some cases, thePCSK9-binding polypeptide formulations can be stored in containers, suchas suitable storage bags (e.g., as manufactured by Sartorius (Gottingen,DE)) or in polycarbonate carboys. Once the pharmaceutical formulationhas been formulated, it can also be stored in pre-filled syringes (PFS;such as 2.25 mL PFS’s) as a solution or suspension in a ready-to-useform, as well as in glass vials (such as 5 cc glass vials).

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

Ultrafiltration/Diafiltration of PCSK9-binding Polypeptide FormulationsComprising N-Acetyl Arginine

PCSK9-binding polypeptides can be formulated to about 210 g/L in thedisclosed UF/DF processes. For example, in the disclosed methods, afirst diafiltration (DF1) concentration of 70 g/L (or 35 g/L) isdiafiltered three times and concentrated to 140 g/L. Then, a seconddiafiltration (DF2) concentration of 140 g/L is diafiltered four timesand concentrated to a concentration of 260 g/L. The concentrated pool isthen recovered from the system with formulation flush to a finalconcentration of 210 g/L.

In the disclosed methods, the first ultrafiltration (UF1) concentrationof about 70 mg/mL and about 35 mg/mL are shown in the Examples to nothave a significant effect on HWM (%) in final drug substance (DS).Furthermore, in the disclosed methods, only seven diavolumes of DF isnecessary at the DF stage to ensure that diafiltration is completed.

The disclosed UF/DF process is summarized in Tables 5 and 6

TABLE 5 UF/DF general procedure and operating parameters ProcessDescription Condition General Membrane/Temperature/ Membrane sizingEquilibrium (EQ)/DF buffer NAR formulation buffers Concentrate to atarget DF conc. Concentration 1 (UF1) Transmembrane pressure (TMP) at 18psi Feed cross-flow rate at 300 LMH (liters/m²/hr) 3 diavolumesDiafiltration 1 (DF1) TMP at 18 psi Feed cross-flow rate at 300 LMHConcentrate to a target DF concentration Concentration 2 (UF2) TMP at 18psi Feed cross-flow rate at 300 LMH 4 diavolumes Diafiltration 2 (DF2)TMP at 18 psi Feed cross-flow rate at 300 LMH Concentrate to a targetconcentration Concentration 3 (over-concentrated; OC) TMP Initially at18 psi; control value fully open Feed cross-flow rate at 60 LMHOperating temp. 37° C. 10 minute recirculation Recirculation Feedcross-flow rate at 60 LMH Permeate path closed (no TMP) Recover proteinsolution through low point or retentate port Recovery Chase with bufferthrough retentate port ≥20 L/m² single-pass Cleaning 30 minrecirculation, 20 L/m² Storage ≥20 L/m² single-pass

TABLE 6 UF/DF process operating parameters Step Parameter Unit ofMeasure Target Operating Range Flush solution PW/water for injection(WFI) or DIW n/a Feed flow LMH 300 n/a Flush Feed volume L/m² 20 n/aPermeate volume L/m² >10 n/a Operational control strategy Single passfiltrate open n/a Integrity test solution PW/WFI or DIWW n/a 0.11 m² =14 n/a Integrity Test Diffusion flow mL/min 0.57 m² = 60 1.14 m² = 117Test pressure psig 30 n/a Test time min 10 n/a NWP NWP solution NWPPW/WFI or DIW 8-14, >70% of new membrane NWP n/a LMH/psig n/a Feed flowLMH 300 n/a Membrane Equilibratior n EQ solution NAR solution n/a Feedvolume L/m² 20 n/a Permeate volume L/m² >10 n/a TMP psig 20 n/aOperational control strategy Single pass filtrate open n/a Concentrationtarget g/L 70 n/a Concentration 1 Feed flow LMH ≤300 n/a TMP psig 18 n/aOperating temp. °C 20 ±5.0 Diafiltration buffer NAR solution n/aDiafiltration 1 Number of diavolumes number 3 n/a Feed flow LMH ≤300 n/aTMP psig 18 ± 5.0 Operating temp. °C 20 ± 5.0 Concentration target g/L140 n/a Concentration 2 Feed flow LMH ≤300 n/a TMP psig 18 ± 5.0Operating temp. °C 20 ± 5.0 Diafiltration buffer NAR solution n/aDiafiltration 2 Number of diavolumes number 4 n/a Feed flow LMH ≤300 n/aTMP psig 18 n/a Operating temp. °C 20 n/a Concentration target g/L 260n/a Concentration 3 Feed flow LMH ≤60 n/a TMP psig Initially at 18. AsTMP valve becomes fully open, TMP is no longer controlled n/a Operatingtemp. °C 37 ± 2.0 Product Recovery Flow mode description Total recyclefiltrate closed n/a Flow rate LMH 60 n/a Low pressure recirculationRecirculation time minutes 10 n/a concentration target g/L 240 n/aRecovery buffer NAR solution n/a Product Recovery Volume of flushthrough retentate Retentate tank Skid hold-up 1 n/a temperature afterrecovery °C 20 ± 2.0 Membrane Cleaning Flow mode Single pass filtrateopen n/a Cleaning solution 0.5 M NaOH n/a Initial flush Feed volume L/m²20 n/a Permeate volume L/m² >10 n/a Feed Flow LMH 300 n/a TMP psig 20n/a Flow mode Total recycle filtrate open n/a Cleaning solution 0.5 MNaOH n/a Recirculation Recirculation Time minutes 30 n/a RecirculationVolume L/m² 20 n/a Feed Flow LMH 300 n/a TMP psig 20 n/a Flow modeSingle pass filtrate open Storage solution 0.1 M NaOH Storage Feedvolume L/m² 20 Permeate volume L/m² >10 Feed flow LMH 300 TMP psig 20

Thus, disclosed herein is a method of formulating a PCSK9-bindingpolypeptide, such as a PCSK9-binding polypeptide that blocks binding ofPCSK9 to LDLR, comprising

-   a. a first concentration step, wherein the polypeptide in a first    solution is concentrated;-   b. a first solution exchange step, wherein the concentrated    polypeptide in the first solution is exchanged into a second    solution comprising N-acetyl arginine using diafiltration;-   c. a second concentration step, wherein the polypeptide in the    second solution is concentrated;-   d. a second solution exchange step, wherein the polypeptide in the    concentrated second solution is exchanged into a third solution    comprising N-acetyl arginine using diafiltration; and-   e. a third concentration step, wherein the polypeptide in the third    solution is concentrated;

Before the third concentration step, the temperature of the solutioncomprising the polypeptide can be increased from about 25° C. to about37° C., such as about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,and about 37° C. Also, the first solution exchange step can beaccomplished using at least three diavolumes of the second solution; insome cases, additional diavolumes can be used, such as four, five, orsix diavolumes. The second solution exchange step can be accomplishedusing at least four diavolumes of the third solution; however,additional diavolumes can be used, include five, six, or sevendiavolumes. The initial concentration of the PCSK9-binding polypeptidecan be about 11 mg/mL or less, such as less than 1 mg, or about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or about 11 mg/mL. Additionally, the PCSK9-bindingpolypeptide concentration can be increased from about 3- to about7-fold, such as about 3-, 4-, 5-, 6-, or about 7-fold. For example, theincreased concentration of the polypeptide is from about 35 mg/mL toabout 70 mg/mL or more, such as about 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, or about 70 mg/mL or more. In thesecond concentration step, the PCSK9-binding polypeptide concentrationis increased from about 2- to 4-fold from the first concentration step(such as from about 2-, 3-, or about 4-fold), such as to about 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, 200, 250, or about 300 mg/mL. In the thirdconcentration step, the PCSK9-binding polypeptide concentration can beincreased from about 1.5-to about 2-fold from the second concentrationstep, such as to about 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,190, 200, 250, or about 300 mg/mL, such as about 260 mg/mL. ThePCSK9-binding polypeptide can therefore have a final concentration thatis at least about 19-20-fold more concentrated than the initialconcentration of the therapeutic polypeptide, such as about 210 mg/mL.The concentration steps can comprise fed-batch ultrafiltration;furthermore, the second solution and the third solution can beidentical.

The second or third solution comprising N-acetyl arginine (e.g., “NARsolution”) can comprise an arginine salt and a buffer, wherein, forexample, the N-acetyl arginine is present at a concentration of about 25mM to about 230 mM, such as about 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210,215, 220, 225, or about 230 mM; the arginine salt is Arg HCl, Argacetate, or Arg glutamate and is present at a concentration of about 25mM to about 150 mM, such as about 25, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, or about 150 mM; and the buffer is a sodium acetate buffer at aconcentration of about 5 mM to about 30 mM, such as about 5, 10, 15, 20,25, or about 30 mM. In other sub-aspects, the N-acetyl arginine ispresent at a concentration of about 140 to about 170 mM; the Arg HCl,Arg acetate, or Arg glutamate is present at a concentration of about 63to about 70 mM (such as about 63, 64, 65, 66, 67, 68, 69, or about 70mM) and the sodium acetate buffer is present at a concentration of about10 mM. For example, the N-acetyl arginine can be present at aconcentration of about 140 mM, the Arg HCl, Arg acetate, or Argglutamate is present at a concentration of about 63 mM, the sodiumacetate buffer is present at a concentration of about 10 mM. In furthersub-aspects, the N-acetyl arginine is present at a concentration ofabout 155 mM, the Arg HCl, Arg acetate, or Arg glutamate is present at aconcentration of about 70 mM, the sodium acetate buffer is present at aconcentration of about 10 mM. In yet another example, the N-acetylarginine is present at a concentration of about 170 mM, the Arg HCl, Argacetate, or Arg glutamate is present at a concentration of about 63 mM,the sodium acetate buffer is present at a concentration of about 10 mM.

Furthermore, the compositions (including the NAR solutions) can furthercomprise proline, wherein the proline is present at a concentration ofabout 50 mM to about 150 mM, such as about 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or about150 mM. The second or third solution can have a pH from about 4.8 toabout 6.9, such as about 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or about6.9, such as 5.3, 5.4, or 5.5. In the first and second solution exchangesteps, a diafiltration membrane can be used having at least onecharacteristic selected from the group consisting of:

-   a. mesh openings that are greater than about 350 µm but less than or    equal to about 500 µm, such as about 350, 360, 370, 380, 390, 400,    410, 420, 430, 440, 450, 460, 470, 480, 490, or about 500 µm;-   b. an open area that is greater than about 32% but less than or    equal to about 36% (such as about 32, 33, 34, 35, or about 36%) of    the membrane area;-   c. a mesh count of less than about 16.2 n/cm but greater than or    equal to about 12.2 n/cm, such as about 16.2, 16, 15.8, 15.6, 15.4,    15.2, 15, 14.8, 14.6, 14.4, 14.2, 14, 13.8, 13.6, 13.4, 13.2, 13,    12.8, 12.6, 12.4 or about 12.2 n/cm;-   d. a wire diameter that is greater than about 270 µm but less than    or equal to about 340 µm, such as about 270, 280, 290, 300, 310,    320, 330, or 340 µm;-   e. a basis weight that is greater than about 160 g/m² but less than    or equal to 180 g/m², such as about 160, 161, 162, 163, 164, 165,    166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,    179, or 180 g/m²;-   f. a thickness greater than about 515 µm but less than or equal to    about 610 µm;-   g. a membrane load of greater than about 1138.1 g/m² but less than    or equal to about 1919.3 g/m²; and-   h. a maximum feed pressure of about 60 psi.

Furthermore, surfactant can added to the third solution after beingconcentrated, such as polyoxyethylenesorbitan monooleate (polysorbate 80or polysorbate 20), polyoxyethylene-polyoxypropylene block copolymer(Poloxamers such as Pluronic® F-68 and other Pluronics®), Sorbitan alkylesters (Spans®) Polyethylene glycol octylphenyl ethers (Triton X-100),Polyethylene glycol alkyl ethers (Brij), Polypropylene glycol alkylethers, Glucoside alkyl ethers, and D-α-tocopherol polyethylene glycolsuccinate (vitamin E TPGS). In some cases, the surfactant ispolyoxyethylene-polyoxypropylene block copolymer (Pluronic® F-68) orD-α-tocopherol polyethylene glycol succinate (vitamin E TPGS). Thesurfactant can range from a concentration from about 0.0001% to about10% weight per volume (“w/v”) of the formulation, e.g., about 0.0001%,about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%,about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 5%, orabout 10% surfactant (w/v) of the formulation. In certain embodiments,the pharmaceutical formulation comprises polysorbate 80 at aconcentration that ranges from about 0.0001% to about 1% w/v of theformulation. In certain embodiments, the pharmaceutical formulationcomprises polysorbate 80 at a concentration at about 0.01% w/v of theformulation. In other embodiments, the formulation comprises Pluronic®F-68 at a concentration that ranges from about 0.0001% to about 1% w/vof the formulation. In certain embodiments, the pharmaceuticalformulation comprises Pluronic® F-68 at a concentration at about 0.01%w/v of the formulation. In still other embodiments, the formulationcomprises vitamin E TPGS at a concentration that ranges from about0.0001% to about 1% w/v of the formulation. In certain embodiments, thepharmaceutical formulation comprises vitamin E TPGS at a concentrationat about 0.01% w/v of the formulation.

The following Examples section is given solely by way of example and arenot set forth to limit the disclosure or claims in any way.

EXAMPLES

Viscosity measurements were made using a TA Instruments (New Castle, DE)AESR-G2 cone and plate rheometer unless otherwise noted.

Example 1 - Design of Experiment (DOE) Study; Optimization of EvolocumabFormulations Containing N-acetyl Arginine

A Design of Experiment (DOE) study was initiated to optimize evolocumabformulations containing N-acetyl arginine. Eleven formulations weretested for viscosity, pre-filled syringe extrusion force, stability, pH,and osmolality. Data on the initial formulation parameters are shown inTable 7.

TABLE 7 Evolocumab NAR design of experiment, time zero data # pH targetTarget [evolocumab] (mg/mL) Target [NAR] Surfactant Target [Acetate] pHOsmolality [evolocumab] (mg/mL) viscosity @ 1000/s (cP) E force 10 sextrusion time (N) E force 12 s extrusion time (N) E force 20 sextrusion time (N) 1 5 190 140 0.05% F68 30 4.94 257 192 29.5 34 28 17 25 210 140 0.05% TPGS 30 4.94 261 204 42.7 47 38 23 3 5.4 190 140 0.01%PS80 10 5.35 243 192 26.8 29 24 15 4 5.4 210 140 0.05% F68 10 5.36 254207 49.0 51 41 25 5 5.2 200 155 0.01% PS 80 20 5.11 265 198 33.5 37 3019 6 5.2 200 155 0.05% F68 20 5.10 261 197 34.6 38 31 19 7 5.2 200 1550.05% TPGS 20 5.10 266 201 32.3 38 31 19 8 5 210 170 0.01% PS 80 30 4.99299 213 45.2 47 38 23 9 5 210 170 0.05% F68 30 5.01 290 213 46.7 48 3924 10 5 210 170 0.05% TPGS 30 4.98 292 214 49.4 49 40 25 11 5.4 190 1700.05% TPGS 10 5.34 286 189 24.3 29 24 15

Acetate concentration was minimized to reduce aggregation rate based onemonth 40° C. data shown in FIG. 1 . Proline concentration was increasedto 120 mM to make the formulation isotonic. N-acetyl arginine (155 mM)was selected to balance between viscosity reduction and thecrystallization of N-acetyl arginine seen at 170 mM at 4° C. Data alsoindicated that varying surfactant between polysorbate-80 (TWEEN® 80;polyoxyethylenesorbitan monooleate), Pluronic® F-68(polyoxyethylene-polyoxypropylene block copolymer), and Vitamin E TPGS(D-α-Tocopherol polyethylene glycol succinate) did not lead to asignificant difference in viscosity or stability.

Based on these data, a formulation of 10 mM acetate, 155 mM N-acetylarginine, 120 mM proline, 0.01% polysorbate-80, pH 5.4 was found to besuitable for a reduced viscosity formulation of evolocumab at highconcentrations (e.g., 190-210 mg/mL). Data presented in Table 8indicated that this formulation is isotonic and has a low viscosity.

TABLE 8 Time zero data for 10 mM acetate, 155 mM N-acetyl arginine, 120mM proline, 0.01% polysobate-80, pH 5.4 at 190-210 mg/mL evolocumabformulations Formulation [evolocumab] pH Osmolality Viscosity10/155/120 - 0.01% PS-80 - 190 mg/mL 188 5.38 290 22.0 10/155/120 -0.01% PS-80 - 200 mg/mL 201 5.40 295 34.5 10/155/120 - 0.01% PS-80 - 210mg/mL 214 5.43 298 51.4

Example 2 - DOE Study to Assess the Impact of Excipient Concentration,pH, and Buffer

Examples 2-4 are directed to formulations that were optimized to reduceviscosity as much as possible while minimizing any impact toaggregation, deamidation, and other stability indicating factors.

N-acetyl arginine (NAR) was selected based on its superior effect inreducing viscosity in excipient screening studies (see for example,Example 1). The concentration of NAR was limited by its solubility at2-8° C. A NAR concentration of 155 mM in diafiltration (DF) buffer wasselected based on stability studies showing no crystallization of NAR atconcentrations as high as 175 mM. Arginine HCl (70 Mm) was added to theDF buffer to achieve an isotonic drug product formulation with thelowest viscosity and to improve the solubility of NAR. The formulationpH of 5.4 was selected to minimize aggregation and deamidation ofevolocumab. Buffer and surfactant (acetate/polysorbate-80) showedminimal effect on evolocumab formulation viscosity and stability.

Three main studies were performed to study the stability of evolocumabin NAR/arginine HCl formulations. They were:

-   1. A DOE study to assess the impact of excipient concentration, pH,    and buffer (acetate vs. glutamate) (Example 2).-   2. A broad pH study which was designed to assess the impact of pH    (5.1 - 6.9) on viscosity/stability (Example 3).-   3. A scale down study to assess the stability of top formulation    candidates including the effects of drug product manufacturing unit    operations (Example 4).

Results

A viscosity target of ≤ 50 cP was established as a target. Formulationviscosity screening indicated that NAR was the most effective excipientto reduce viscosity. However, due to limited NAR solubility, acombination of NAR and arginine HCl was used to achieve an isotonicformulation and to meet the objective of a 210 mg/mL evolocumabformulation with a viscosity less than or equal to 50 cP. A summary ofselected formulations that were evaluated during screening is shown inFIG. 2 .

A DOE study was designed to assess the impact of NAR/arginine HClconcentration, pH, and buffer species on evolocumab stability andviscosity at 210 mg/mL. Formulation variables for each sample are shownin Table 9. The study design included NAR concentrations from 155-175mM, arginine HCl concentrations from 50-100 mM, and pH from 4.8 to 5.4.The study also included a comparison between acetate and glutamatebuffers at 10 mM. The study included a 140 mg/mL proline formulationcontrol which was prepared and filled in PFS from the same lot ofstarting material as NAR/Arg HCl samples. Samples were sterile filteredusing 0.2 µm PVDF filters and hand-filled in glass PFS at a fill volumeof ~2.0 mL.

TABLE 9 DOE study design pH NAR (mM) Arg HCl (mM) Buffer PS-80Evolocumab (mg/mL) Sample code 4.8 155 50 10 mM Acetate 0.01% 210 DOE 1-10/155/50 pH 4.8 5.4 155 50 10 mM Glutamate 0.01% 210 DOE 2 - 10/155/50pH 5.4 4.8 175 50 10 mM Glutamate 0.01% 210 DOE 3 - 10/175/50 pH 4.8 5.4175 50 10 mM Acetate 0.01% 210 DOE 4 - 10/175/50 pH 5.4 5.1 165 75 10 mMAcetate 0.01% 210 DOE 5 - 10/165/75 pH 5.1 5.1 165 75 10 mM Glutamate0.01% 210 DOE 6 - 10/165/75 pH 5.1 4.8 155 100 10 mM Glutamate 0.01% 210DOE 7 - 10/155/100 pH 4.8 5.4 155 100 10 mM Acetate 0.01% 210 DOE 8 -10/155/100 pH 5.4 4.8 175 100 10 mM Acetate 0.01% 210 DOE 9 - 10/175/100pH 4.8 5.4 175 100 10 mM Glutamate 0.01% 210 DOE 10 - 10/175/100 pH 5.45.0 0 0 20 mM Acetate 0.01% 140 140 mg/mL Proline Control

Data are tabulated for pH, concentration, osmolality, and viscosity inTable 10. pH values were observed to be close to targets for allsamples. Osmolality in the study covered a range from 256-392 mOsm/kg.Viscosity appeared to show an inconsistent trend towards lower viscosityat higher pH.

TABLE 10 pH, concentration, osmolality, and viscosity data from DOESample pH Conc. mOsm/kg Viscosity (cP @ 1000 sec⁻¹, 25° C.) 1 -10/155/50 pH 4.8 4.83 209.0 256 45.3 2 - 10/155/50 pH 5.4 5.29 209.0 27143.8 3 - 10/175/50 pH 4.8 4.81 210.6 301 46.6 4 - 10/175/50 pH 5.4 5.31212.9 297 41.0 5 - 10/165/75 pH 5.1 5.07 211.9 326 48.2 6 - 10/165/75 pH5.1 5.03 209.8 340 47.5 7 - 10/155/100 pH 4.8 4.80 208.5 369 44.9 8 -10/155/100 pH 5.4 5.29 210.3 366 38.8 9 - 10/175/100 pH 4.8 4.84 208.7376 43.9 10 - 10/175/100 pH 5.4 5.33 209.2 392 36.6

Analysis of size-exclusion high pressure liquid chromatography (SE-HPLC)data using JMP statistical analysis software (SAS; Cary, NC; FIGS. 3Aand 3B) revealed significant effects of pH and arginine HClconcentration on loss of percent main peak at 40° C. for 1 month (FIG.3A). Lower pH and higher arginine HCl concentration caused greater lossof main peak primarily due to an increase in aggregate peak and to alesser extent, oligomer peak. Both peaks combined were categorized ashigh molecular weight (HMW) species. FIG. 4 shows the effects of pH andarginine HCl on percent of HMW species for the 1 month, 40° C. timepoint. There were significantly higher levels of percent of HMW speciesseen in pH 4.8 samples compared to pH 5.4 samples. Also, the effect ofhigher arginine HCl concentration leading to higher percent of HMWspecies was significantly more pronounced at pH 4.8 compared to pH 5.4.

FIGS. 5A-5C show SE-HPLC chromatograms of selected pH 4.8 and pH 5.4samples compared to 140 mg/mL proline control at 5° C. for 6 months, 25°C. for three months, and 40° C. for 1 month, respectively. Thechromatograms show the trend of increasing levels of aggregate at pH 4.8relative to pH 5.4. The chromatograms also indicate that the degradationprofile was comparable for 210 mg/mL NAR/Arg HCl samples compared to the140 mg/mL proline control despite faster rate of aggregation observedfor 210 mg/mL NAR/Arg HCl samples.

Despite pH and Arg HCl concentration-dependent differences seen at 40°C., all formulations studied in this experiment showed comparablepercent HMW species for up to 6 months at 5° C. and up to 1 month at 25°C. (FIGS. 6A, 6B).

Tryptic peptide mapping with liquid chromatography-mass spectrometry(LC-MS) analysis was performed on selected samples Peptide mapHPLC-ultraviolet (UV) chromatograms were visually compared to prolineevolocumab formulation controls and evolocumab reference standard. Nonew peaks were observed in NAR/Arg HCl samples relative to controls(FIG. 8 ). In addition, analysis of chemical modifications detected bymass spectrometry showed no significant changes between NAR/Arg HClsamples and controls. Relative quantitation was performed andpercentages of N55 and N33 deamidation as well as M246 and M422oxidation are shown in Table 11. Slightly higher levels of N55deamidation were seen in pH 5.4 NAR/Arg HCl samples relative to pH 5.1NAR/Arg HCl and pH 5.0 proline samples, consistent with the increase inacidic peak seen by CEX. N33 (a potential deamidation site in thecomplementary determining region (CDR)) did not appear to show asignificant increase in deamidation in the pH range and conditionsassessed. Oxidation rates for both M246 and M422 were higher for 140mg/mL proline control samples compared to 210 mg/mL NAR/Arg HCl samplesfollowing incubation at ASC.

TABLE 11 Deamidation and oxidation of evolocumab (selected samples)Sample % N55 Deamidation % N33 Deamidation % M246 Oxidation % M422Oxidation 10/165/75 Acetate pH 5.1 5° C. 3 M 11.6 0.8 5.4 3.7 10/165/75Acetate pH 5.1 25° C. 3 M 11.8 0.7 6.0 3.8 10/165/75 Acetate pH 5.1 40°C. 1 M 13.6 0.9 8.4 5.1 10/155/100 Acetate pH 5.4 5° C. 3 M 12.3 0.9 5.43.7 10/155/100 Acetate pH 5.4 25° C. 3 M 12.1 0.6 6.1 3.9 10/155/100Acetate pH 5.4 40° C. 1 M 15.2 0.8 8.4 5.2 140 mg/mL Proline Control pH5.0 5° C. 3 M 10.6 0.6 5.6 3.7 140 mg/mL Proline Control pH 5.0 25° C. 3M 10.8 0.7 6.7 3.8 140 mg/mL Proline Control pH 5.0 40° C. 1 M 11.8 0.711.0 6.5

Sub-visible particles by light obscuration liquid borne particlecounting and micro-flow imaging (MFI) showed no significant trendscorrelating with the formulation variables studied (FIGS. 9A-9C and FIG.10 ). Sub-visible particle counts were comparable between 210 mg/mLNAR/Arg HCl samples and the 140 mg/mL proline control.

FIG. 11 compiles viscosity data from this study at time zero, threemonth, and six month time points. The data indicate that viscosityremained stable for up to six months at 5° C. and 25° C. for allformulations. 40° C. samples showed a pH-dependent increase in viscositywhich was correlated to the increase in percent HMW species seen bySE-HPLC at 40° C. (FIG. 4 ).

Observations

An approximately isotonic formulation (~300 mOsm/kg) was achieved at thestudy midpoint formulation (10 mM buffer, 165 mM NAR, 75 mM arginineHCl)

Increasing pH lead to a decrease in percent aggregate followingaccelerated storage as observed using size exclusion chromatography(SEC) as well as an increase in deamidation with higher percent acidicspecies as detected by cation exchange.

Arginine HCl concentration had a greater impact on stability (lead toincreased aggregation at 25° C. and 40° C.) at pH 4.8 and its effect wasminimized at pH 5.4.

Acetate and glutamate are comparable in their effects on stability andviscosity.

No new peaks were observed for NAR/Arg HCl vs. proline controls bypeptide mapping of selected samples for time points up to three monthsat 5° C. and 25° C. and one month at 40° C.

Viscosity remains stable up to six months at 5° C. and 25° C.

NAR remains soluble in formulations at 5° C. with DF bufferconcentrations up to 175 mM with Arg HCl concentrations between 50-100mM.

Example 3 - pH Study

A pH study was designed to further investigate the effect of pH across abroader range. The study included formulations with a target pH rangefrom 5.1-6.9. pH and buffer used for each sample is listed in Table 12.All samples formulated at 210 mg/mL with 10 mM buffer, 165 mM NAR, 75 mMArginine HCl, 0.01% Polysorbate-80. Samples were sterile-filtered using0.2 µm PVDF filters and hand-filled in glass PFS at a fill volume of 2.0mL.

TABLE 12 pH and buffer variables for evolocumab (210 mg/mL) formulationshaving 165 mM NAR,75 mM Arg HCl, and 0.01% (w/v) polysorbate-80 SamplepH Buffer 1 5.1 Acetate 2 5.4 Acetate 3 5.7 Acetate 4 6.0 Histidine 56.3 Histidine 6 6.6 Phosphate 7 6.9 Phosphate

Results

The pH values for each sample at time zero and over three months at 40°C. are shown in FIG. 12 .

FIGS. 13A-13C show the effect of pH on percent HMW species (percentoligomer + percent aggregate) by SE-HPLC for up to three months at 4°C., 25° C., and 40° C., respectively. The 5° C. and 25° C. data showedminimal differentiation between formulations of varying pH. The mostsignificant difference correlated with pH was higher levels of percentHMW species from time zero for pH 6.6 and pH 6.9 formulations. FIG. 14shows a plot of time zero oligomer levels vs pH. A slight upward trendin oligomer was observed with increasing pH with a sharper increaseobserved above pH 6.3. Oligomer levels were minimized at lower pH.

As seen previously in the DOE study (Example 2), percent HMW speciesincreased over time at 40° C. for each pH studied, with lower pHcorrelated with higher initial rates of increase.

From a comparison of SE-HPLC chromatograms at the 40° C., three-monthtime point, increased levels of aggregate peak arose with decreasing pH(FIG. 15 ).

FIGS. 16A-16C show the effect of pH on percent main peak by CEX-HPLC forup to six months at 5° C. (FIG. 16A), 25° C. (FIG. 16B), and 40° C.(FIG. 16C). At 5° C., six month time point, there was no significantchange in percent main peak for samples with pH ≤ 6.0, while there wasan observed decrease in percent main peak for samples with pH ≥ 6.3(FIG. 16A). CEX-HPLC data at ASC in FIG. 16B and FIG. 16C indicated thatpercent main peak decreased with increasing pH and that the rate ofdecrease significantly accelerated at pH > 6.0.

FIG. 17 shows that the pH dependent decrease in CEX percent main peak at40° C. was due to an increase in percent acidic species.

A comparison of CEX-HPLC chromatograms at the 25° C., three-month timepoint can be seen in FIG. 18 . Percent acidic peak grew with increasingpH and there was a significant change in the chromatographic profile ofsamples having a pH greater than 6.3.

Tryptic peptide mapping with liquid chromatography-mass spectrometry(LC-MS) analysis was performed on samples after storage for 1 month at40° C. Peptide map HPLC-UV chromatograms (FIG. 19 ) were visuallycompared to reference standard and across the pH range. In addition,chemical modification analysis by mass spectrometry showed nosignificant changes correlated with pH except for N55 and N33deamidation. Relative quantitation levels of N55 and N33 deamidation aswell as M246 and M422 oxidation are shown in Table 13. Increases inlevels of percent N55 deamidation and percent N33 deamidation correlatedwith increased levels of percent acidic peak seen in CEX-HPLC data withincreasing pH. There did not appear to be a significant effect of pH onoxidation levels for M246 or M422.

TABLE 13 Deamidation and oxidation of evolocumab Sample % N55Deamidation % N33 Deamidation % M246 Oxidation % M422 Oxidation pH 5.1 -5° C. 4 M 10.7 0.7 5.8 1.5 pH 5.1 - 40° C. 1 M 13.7 1.1 7.1 1.9 pH 5.4 -40° C. 1 M 15.5 0.9 6.5 1.5 pH 5.7 - 40° C. 1 M 19.6 1.3 6.9 1.6 pH6.0 - 40° C. 1 M 24.8 2.0 6.3 1.5 pH 6.3 - 40° C. 1 M 31.9 2.4 5.8 1.1pH 6.6 - 40° C. 1 M 53.3 4.9 6.7 1.3 pH 6.9 - 40° C. 1 M 70.5 10.0 6.91.4

Bioassay results for one month 40° C. samples is plotted in FIG. 20alongside percent initial main peak (CEX-HPLC) and percent N55 andpercent N33 (not deamidated).

Sub-visible particles by light obscuration liquid borne particlecounting (FIGS. 21A-21C) and MFI (FIG. 22 ) showed no significant trendsthat correlated with pH.

Data in FIG. 23 shows that pH had a minimal effect on fragmentation orother degradation as measured by reduced capillary electrophoresis -sodium dodecyl sulfate (rCE-SDS) analysis for up to six months at 25° C.Percent pre light chain plus light chain + heavy chain (pre LC + LC +HC) in FIG. 23 shows a slight decrease at the edges of the pH range.Lower pH samples contained slightly higher percent mid molecular weight(MMW) species while higher pH samples contained slightly higher percentHMW species. Percent low molecular weight (LMW) species is comparableacross the pH range.

Finally, as shown in FIG. 24 , there is a good linear relationshipbetween viscosity and pH, with a decrease of 6.6 cP per pH unit.

Observations

Increasing pH led to (1) decreased aggregation rate at 40° C., (2)higher initial levels of oligomer, (3) increased rate of deamidation at25° C. and 40° C., and (4) lower viscosity. pH did not appear to have asignificant effect on visible or sub-visible particles, nor did pHappear to have a significant effect on fragmentation or otherdegradation as measured by rCE-SDS.

Example 4 - Scale Down Study

Three formulations were subjected to commercial manufacturability andstability assessment. Formulation candidates were subjected to variousunit operations, simulating commercial manufacturing prior to beingplaced on stability

Results

Physical properties were determined for each formulation drug substance.

pH was determined to be ~ 0.17 higher than the DF buffer pH for eachformulation. DF buffer for formulation 3 was 0.14 above target whichresulted in a DS pH which was 0.31 above target.

SE-HPLC (percent HMW species) results for drug product stability at 5°C., 25° C., and 40° C. are shown in FIGS. 25A-25C. Rates of increase inpercent HMW species were comparable for all three samples at 5° C. and25° C. while the rate of aggregation was slightly higher for sample 1(having a lower pH) at 40° C.

CEX-HPLC data are shown in FIGS. 26A-26C and show the expected pH andtemperature dependent increases in percent acidic peak that wereobserved in previous studies (see previous Examples).

rCE-SDS data shown in FIG. 27 shows comparable levels of percent PreLC +HC + LC for each of the scale down formulations at all temperature andtime points tested.

Turbidity levels did not significantly change for up to four months at5° C. and 25° C. but did increase after 4 months at 40° C.

Scale down sample concentrations were adjusted to 200, 210, and 220mg/mL for each formulation. Samples were tested by the m-VROC™ rheometer(RheoSense; San Ramon, CA) at shear rates up to 90,000 sec⁻¹ and attemperatures from 18° C. to 28° C. The data in FIG. 28 spanned aviscosity range from 22- 52 cP and illustrated the impacts of proteinconcentration, temperature, and formulation variation on viscosity.

Observations

Levels of percent HMW species and aggregation rates by SE-HPLC wereconsistent with those observed in previous studies (e.g., see previousExamples).

Levels of percent main, acidic, and basic peaks and rates of change byCEX-HPLC were consistent with those observed in previous studies.

rCE-SDS data showed no significant fragmentation or other degradation upto three months at RSC and ASC

Example 5 - Ultrafiltration/Diafiltration of Evolocumab in FormulationsComprising N-Acetyl Arginine Materials and Methods

The small-scale ultrafiltration/diafiltration (UF/DF) developmentexperiments used Pellicon® 3 Cassette with Ultracel PLCTK Membrane, 30kD molecular weight cut-off (D Screen, 0.11 m²; EMD Millipore;Billerica, MA). The experiments were performed on a Tangential FlowFiltration (TFF) Process System (PendoTECH; Princeton, NJ). Theexperiments were performed at room temperature (22.2±2° C.).

The UF/DF development experiments were performed at small scale toevaluate three NAR formulation buffers with different pH values andcompared their permeate flux data over concentration. Two DF steps wereused in order to save cost on decreasing consumption of NAR. Additionalexperiments were performed in the UF1/DF1 step to evaluate two targetconcentrations (35 mg/mL and 70 mg/mL) on the high molecular weightsformulation during the UF/DF process. Other UF/DF operating parameterswere not evaluated. General procedure of UF/DF experiments is describedin Table 14.

TABLE 14 UF/DF general procedure and operating parameters ProcessDescription Condition Evaluated General Membrane/Temperature/ Membranesizing N Equilibrium (EQ)/DF buffer NAR formulation buffers YConcentrate to a target DF conc. Y Concentration 1 (UF1) Transmembranepressure (TMP) at 18 psi Feed cross-flow rate at 300 LMH (liters/m²/hr)N 3 diavolumes Y Diafiltration 1 (DF1) TMP at 18 psi Feed cross-flowrate at 300 LMH N Concentrate to a target DF concentration YConcentration 2 (UF2) TMP at 18 psi Feed cross-flow rate at 300 LMH N 4diavolumes Y Diafiltration 2 (DF2) TMP at 18 psi Feed cross-flow rate at300 LMH N Concentrate to a target concentration Y Concentration 3(over-concentrated; OC) TMP Initially at 18 psi; control value fullyopen Feed cross-flow rate at 60 LMH Operating temp. 37° C. NRecirculation 10 minute recirculation Feed cross-flow rate at 60 LMHPermeate path closed (no TMP) N Recovery Recover protein solutionthrough low point or retentate port Chase with buffer through retentateport N Cleaning ≥20 L/m² single-pass 30 min recirculation, 20 L/m² NStorage ≥20 L/m² single-pass N

A₂₈₀ measurements were performed using a variable path lengthspectrophotometer (SoloVPE system; SoloVPE; Bridgewater, NJ) with anextinction coefficient of 1.5 (cm)⁻¹ (g/L)⁻¹.

Analytical methods used to evaluate product pool quality includedSE-HPLC, rCE-SDS, and CEX-HPLC.

NAR: Ac-Arg-OH, Biochem Catalog number- E-1025

NAR Formulation Buffer Study Results

This experiment evaluated three NAR formulation buffers in the UF/DFprocess. The UF/DF process followed the general guideline: (1)evolocumab was concentrated to 70 mg/mL through fed-batch concentration(UF1) and diafiltrated to NAR formulation buffer with 3 diavolumes(DF1); (2) the protein was further concentrated to 140 mg/mL (UF2) anddiafiltrated to NAR formulation buffer with 4 diavolumes (DF2); (3) theprotein was over-concentrated to target ~260 mg/mL and recovered fromthe system at 37° C. The protein loading/member area was 1468 g/m² forNAR pH 5.4 and 800 g/m² for NAR pH 5.2 and 5.6.

The flux data was plotted over concentration and compared across all NARformulation buffers. In addition, the excipient levels were analyzed bysampling from each diafiltration step to study diafiltration performanceand determine the minimal diavolumes.

Results and Observations

The flux data from the UF/DF study was used to generate the filtrateflux vs. concentration plot shown in FIG. 29 . The flux profiles werecomparable across the three NAR formulation buffers. The flux increasedwith protein being buffer exchanged to NAR formulation buffer (DF1 andDF2), but decreased significantly with increase in protein concentration(UF1, UF2, and OC). The total process time for UF/DF was about 20 hoursin NAR pH 5.4 and 10 hours in NAR pH 5.2 and 5.6 due to differentmembrane areas. The similar flux profile results showed that the NARformulation buffer had no significant effect on the process flux. Thesummary of evolocumab drug substance (DS) small scale UF/DF studies areshown in Table 15.

Samples were taken at the diafiltration steps (DF1 and DF2) at eachdiavolume (1 to 7 DVs) to test diafiltration performance and determineminimal diavolumes. The representative sample analyses in Table 16 werefrom evolocumab NAR pH 5.4 UF/DF study. The results showed that afterfive diavolumes, the diafiltration to NAR pH 5.4 was essentiallycomplete.

TABLE 15 Summary of evolocumab DS Small scale UF/DF studies Variable pH5.2 pH 5.4 pH 5.6 Feed materials evolocumab HMP VFP 11 mg/mL Formulationbuffer 10 mM Acetate 10 mM Acetate 10 mM Acetate 140 mM NAR 155 mM NAR170 mM NAR 63 mM Arg HCl 70 mM Arg HCl 63 mM Arg HCl Membrane load(g/m²) 800 1467.5 800 OC (g/L) 262 270 268 DS concentration (g/L) 226217 222 DS viscosity cP @1000s-1 77.4 49.6 52.3

TABLE 16 Excipient levels at each DF step in evolocumab UF/DF NAR pH 5.4Sample Acetate (mM) Arginine (mM) NAR (mM) Na (mM) Cl (mM) Tris (mM) DF091.243 NA NA 155.97 99.9 1.29 DF1-1DV 38.271 34.193 115.805 25.94 69.80.21 DF1-2DV 20.435 52.299 153.178 26.78 60.4 0.22 DF1-3DV 13.903 44.951163.453 9.88 56.8 0.08 DF2-4DV 11.354 46.28 167.072 6.08 56.4 0.05DF2-5DV 10.287 45.819 157.11 4.69 55.5 0.04 DF2-6DV 10.354 46.283166.749 3.80 61.7 0.03 DF2-7DV 10.134 46.488 162.536 4.86 58.1 0.04

Example 6 - Monitoring DS Concentration and HMW Formation During UF/DFof Evolocumab Formulations

The purpose of the experiment was to evaluate the target concentrationon Fed-Batch Concentration/Diafiltration (UF1/DF1), and to determine thetarget concentration to minimize the HMW formation during UF/DFoperation. The UF/DF process followed the general guidelines listed inTable17. Evolocumab (11-mg/mL) was concentrated to 35 mg/mL or 70 mg/mLthrough Fed-batch concentration (UF1). The protein loading/member areawas 800 g/m² and the NAR formulation buffer included 10 mM Acetate, 155mM N-Acetyl arginine, 70mM arginine HCl, pH 5.3.

In addition, from each UF/DF step, protein samples were taken forproduct pool quality analysis in order to evaluate the UF/DF operation.The product pool quality data were used to study the effect of differentUF1/DF1 product concentration on HMW formation.

Materials and Methods

Refer to Example 5.

Results and Observations

FIG. 30 shows a plot comparing HMW (%) formation in evolocumab UF/DFprocess with 35 mg/mL and 70 mg/mL in UF1/DF1 (UF/DF-70 and UF/DF-35).Initially, the HMW species (%) in UF/DF-70-UF1 was 0.2 % higher thanUF/DF-35-UF1, but the HMW (%) were comparable after UF1 and the same inthe final DS. The result indicated that the HWM species was reversibleand the target concentration on UF1 does not have significant effect onHWM species formation in final DS. It is recommended that 70 mg/mL bethe target concentration to reduce pool volume.

Example 7 - Pool Hold Studies on Three NAR DS From Small Scale UF/DF

In this example, the stability of three batches of evolocumab formulatedto three NAR formulation buffers was monitored. Ten mL of each DS samplewas held at 2-8° C. At each time point shown in FIGS. 31-A-31F, about 1mL sample was taken out and shipped at 2-8° C. for analytical testing.The values of each bar shown in the graphs of FIGS. 31A-31F are providedin Tables 17A-17F, respectively.

TABLE 17A Evolocumab NAR DS pH 5.2-SE-HPLC T0 T7 T21 T35 T49 T77 % HMW1.4 1.5 1.7 1.7 1.8 1.9 % Main 98.6 98.5 98.3 98.2 98.2 98.1 %LMW 0 0 00.1 0 0

TABLE 17B Evolocumab NAR DS pH 5.2-CE-HPLC T0 T7 T21 T35 T49 T77 % HMW35.8 36 35.4 34.8 34.8 35.3 % Main 56.6 56.4 56.4 56.8 57.1 57.6 %LMW7.6 7.6 8.2 8.5 8.1 7.1

TABLE 17C Evolocumab NAR DS pH 5.4-SE-HPLC T0 T7 T21 T35 T49 T77 % HMW1.5 1.5 1.7 1.8 1.8 1.9 % Main 98.5 98.5 98.3 98.2 98.2 98.1 %LMW 0 0 00 0 0

TABLE 17D Evolocumab NAR DS pH 5.4-CE-HPLC T0 T7 T21 T35 T49 T77 % HMW35.3 35.5 35.1 34.6 34.7 35.6 % Main 57.1 56.6 56.8 57 57.1 57.5 %LMW7.6 8 8.1 8.3 8.1 7

TABLE 17E Evolocumab NAR DS pH 5.6-SE-HPLC T0 T7 T21 T35 T49 T77 % HMW1.7 1.8 2 2.1 2.2 2.2 % Main 98.3 98.2 98 97.9 97.8 97.7 %LMW 0 0 0 0 00.1

TABLE 17F Evolocumab NAR DS pH 5.6-CE-HPLC T0 T7 T21 T35 T49 T77 % HMW36.1 36 35.5 35.2 35.2 36.2 % Main 56.3 56.2 56.2 56.5 56.5 56.7 %LMW7.7 7.8 8.3 8.3 8.3 7.1

Results and Observations

SE-HPLC and CEX results of evolocumab NAR evolocumab at 2-8° C. areshown in FIGS. 36 and 37 . CEX and rCE-SDS results yielded comparableresults. The DS batches generated from NAR pH 5.2, 5.4, and pH 5.6possess 1.4 %, 1.5 %, and 1.7 % HMW species respectively during UF/DFoperation compared to 1.1 % in the feed material. The % of HMW specieswas plotted to compare the evolocumab feed material and NAR DS for 11weeks in FIG. 32 . All three of the formulation batches had HMW (%)species increase by 0.4 %- 0.5 %. These DS batches were stable at leastfor 7 days at 2-8° C.

Example 8 - In-Process Pool Hold Studies on Evolocumab HMP CPD1 NAROverconcentration (260 g/L) at 37° C., 39° C., 42° C., and 45° C.

The stability of the in-process evolocumab overconcentration (260 g/L)at 37° C., 39° C., 42° C., and 45° C. was evaluated. Thirty mL of OCsamples was incubated in a water bath in a room with set controlledtemperatures. At each time point shown in FIGS. 33 and 34 , about 1 mLsample was taken, frozen, and shipped on dry ice for analysis. Viscosityvalues were measured on the OC samples at the higher temperatures andcompared to those at held at 23° C. and 25° C.

Results and Observations

SE-HPLC results showed that evolocumab NAR OC was stable for three hoursat 37° C., 39° C., and 42° C., but not at 45° C. as shown in FIG. 33 .CEX and rCE-SDS results were comparable. FIG. 34 shows that theviscosity of OC decreased when the temperature increased and, whentemperature increased from 37° C. to 42° C., the viscosity dropped by19% (by 14.7 cP).

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including patents, patent applications, articles, books, and treatises,are hereby expressly incorporated by reference in their entirety for anypurpose.

Example 9 - Formulation Robustness DOE Study

A formulation robustness design of experiment (DOE) study was designedto investigate the effect of formulation variables within a specifieddesign space on evolocumab stability. The study variables includedevolocumab concentration, NAR concentration, Arg HCl concentration, andpH. Eighteen formulations listed in Table 18 were sterile-filtered using0.2 µm PVDF filters and filled in 2.0 mL glass pre-filled syringes.Samples were tested by an array of stability indicating analyticalmethods following incubation at various temperatures. All formulationsincluded 10 mM sodium acetate and 0.01% (w/v) polysorbate-80 in additionto the components listed in Table 18.

TABLE 18 Study design Sample Sample code [evolocumab] (mg/mL)Excipients* pH 1 210A54NARRT80 210 140 mM NAR, 50 mM Arg HCl 5.4 2210A54NARRT80 210 140 mM NAR, 50 mM Arg HCl 5.4 3 197A51NAR₁₅₄R₅₅T80 197154 mM NAR, 55 mM Arg HCl 5.1 4 197A51NAR₁₂₆R₄₅T80 197 126 mM NAR, 45 mMArg HCl 5.1 5 223A51NAR₁₅₄R₄₅T80 223 154 mM NAR, 45 mM Arg HCl 5.1 6223A51NAR₁₂₆R₄₅T80 223 126 mM NAR, 45 mM Arg HCl 5.1 7197A57NAR₁₂₆R₅₅T80 197 126 mM NAR, 55 mM Arg HCl 5.7 8197A51NAR₁₂₆R₅₅T80 197 126 mM NAR, 55 mM Arg HCl 5.1 9197A51NAR₁₅₄R₄₅T80 197 154 mM NAR, 45 mM Arg HCl 5.1 10223A51NAR₁₂₆R₅₅T80 223 126 mM NAR, 55 mM Arg HCl 5.1 11223A57NAR₁₂₆R₄₅T80 223 126 mM NAR, 45 mM Arg HCl 5.7 12223A57NAR₁₅₄R₅₅T80 223 154 mM NAR, 55 mM Arg HCl 5.7 13223A57NAR₁₅₄R₄₅T80 223 154 mM NAR, 45 mM Arg HCl 5.7 14197A57NAR₁₂₆R₄₅T80 197 126 mM NAR, 45 mM Arg HCl 5.7 15197A57NAR₁₅₄R₅₅T80 197 154 mM NAR, 55 mM Arg HCl 5.7 16223A57NAR₁₂₆R₅₅T80 223 126 mM NAR, 55 mM Arg HCl 5.7 17197A57NAR₁₅₄R₄₅T80 197 154 mM NAR, 45 mM Arg HCl 5.7 18223A51NAR₁₅₄R₅₅T80 223 154 mM NAR, 55 mM Arg HCl 5.1 *formulationsincluded 10 mM sodium acetate and 0.01% (w/v) polysorbate-80, as notedin text

Table 19 shows the measured evolocumab concentrations, pH values, andexcipient concentrations for each of the study formulations. All areclose to the target levels listed in Table 18.

TABLE 19 Initial drug product formulation data Sample # [evolocumab](mg/mL) pH [NAR] (mM) [Arginine] (mM) 1 209 5.51 145.0 56.7 2 211 5.46143.5 56.2 3 191 5.14 156.7 60.9 4 201 5.17 126.3 46.9 5 226 5.13 160.248.2 6 225 5.13 127.8 47.9 7 198 5.67 135.8 63.1 8 199 5.14 125.1 59.1 9202 5.13 161.8 49.9 10 224 5.12 136.1 64.5 11 220 5.71 132.1 51.5 12 2235.73 161.7 63.3 13 218 5.75 159.7 50.7 14 197 5.76 128.5 50.5 15 1945.79 157.6 61.7 16 227 5.72 141.0 67.3 17 196 5.72 154.2 58.1 18 2265.17 153.6 57.8

Results and Observations

To assess aggregation, SE-HPLC analysis was conducted on theformulations shown in Table 19 after 0-6 months incubation at 4° C.(FIG. 35A), 25° C. (FIG. 35B), and 40° C. (FIGS. 35C and 35D). The dataindicated that rates of increase in % HMW species were similar for allformulations for 4° C. and 25° C. conditions, but a pH-dependent effecton aggregation was observed at the accelerated 40° C. stress condition.Lower pH samples at pH 5.1 were observed to have the fastest rate ofaggregation at 40° C. while samples having a pH 5.7 were observed tohave the slowest rate of aggregation (FIGS. 35C and 35D). Samples at pH5.4 at 40° C. were observed to have intermediate aggregation ratesbetween those of pH 5.1 and 5.7, but these rates were more similar tothe lower aggregation rates observed for samples of pH 5.7 (FIGS. 35Cand 35D).

The effect of pH on increase in % acidic peak and % basic peak over time(up to three months) was examined using CEX-HPLC, which data are shownin FIG. 36A (acidic peak) and 36B (basic peak) for 4° C.; FIG. 36C(acidic peak) and 36D (basic peak) for 25° C.; and FIG. 36E (acidicpeak) and 36F (basic peak) for 40° C. As shown in the data for 25° C.and 40° C. samples (FIGS. 36C-36F), pH was observed to effect % acid andbasic peaks in the formulations. Data for samples with pH 5.7 weretightly grouped with higher rates of increase in % acidic peak whiledata for samples with pH 5.1 showed the lowest rate of increase. The twosamples of pH 5.4 were observed to have degradation rates directlybetween the observed rates of those samples of pH 5.1 and pH 5.7. Trendsin % basic peak changes appeared to be less distinct, but a trend towardhigher % basic peak levels at lower pH were observed from samplesincubated at 40° C.

Fragmentation or other degradation of evolocumab in the formulations forup to three months at either 30° C. or 40° C. was assayed using rCE-SDSand SE-HPLC analyses; FIGS. 37A-37B present the data for samplesincubated at 30° C., and FIGS. 37C-37D show the data for samplesincubated at 40° C. No significant trend in the rCE-SDS data related toformulation composition within the design space of this study wasobserved.

The presence and amounts of subvisible particles were determined for theformulations over time for up to three months as determined by lightobscuration particle counting using HIAC following incubation at 4° C.and 40° C.; these data are shown in FIGS. 38A-38D. (FIG. 38A (greaterthan or equal to 10 µm) and 38B (particles greater than or equal to 25µm) shows the results for samples held at 4° C.; FIG. 38C (greater thanor equal to 10 µm) and 38D (particles greater than or equal to 25 µm)show the results for samples held at 40° C.). From these data, nosignificant trend in subvisible particle counts related to the studiedformulation variables was observed.

From this example, the data demonstrated that within the studied narrowformulation design space, no significant impact to stability duringstorage at 4° C. or 25° C. was observed. The data related to pH at 40°C. suggested that pH effected aggregation rate as detected by SE-HPLC aswell as percent acidic species and to a lesser extent percent basicspecies as detected by CEX-HPLC.

EXEMPLARY EMBODIMENTS

Disclosed herein, are exemplary embodiments of the pharmaceuticalcompositions comprising evolocumab, wherein such compositions compriseN-acetyl arginine and the pharmaceutical composition has a viscosity ofat least less than about 80 cP (measured, for example, by a rheometer,such as TA Instruments (New Castle, DE) AESR-G2 cone and platerheometer). Furthermore, disclosed herein are methods of formulatingtherapeutic polypeptides, such as evolocumab, wherein such compositionscomprise NAR.

Embodiment 1: A pharmaceutical composition comprising

-   a. a PCSK9-binding polypeptide that selected from the group    consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, I189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR; and-   b. N-acetyl arginine,-   wherein the pharmaceutical composition has a viscosity of at least    less than about 80 cP.

Embodiment 2: The pharmaceutical composition of embodiment 1, whereinthe PCSK9-binding polypeptide is a monoclonal antibody the comprises aheavy chain polypeptide comprising the following complementaritydetermining regions (CDRs):

-   a. heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs:7, 8, and 9, respectively; and-   b. light chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs:4, 5, and 6, respectively.

Embodiment 3: The pharmaceutical composition of embodiment 2 or 3,wherein the pharmaceutical composition has a viscosity of at least lessthan about 50 cP.

Embodiment 4: The pharmaceutical composition of embodiment 2 or 3,wherein the pharmaceutical composition has an osmolality of about 250 toabout 400 mOsm/kg.

Embodiment 5: The pharmaceutical composition of embodiment 4, whereinthe pharmaceutical composition has an osmolality of about 300 mOsm/kg.

Embodiment 6: The pharmaceutical composition of embodiment 5, whereinthe pharmaceutical composition is isotonic to a human blood cell.

Embodiment 7: The pharmaceutical composition of embodiment 1, whereinthe PCSK9-binding polypeptide is present at a concentration of about 140mg/mL to about 260 mg/mL.

Embodiment 8: The pharmaceutical composition of embodiment 2, whereinthe PCSK9-binding polypeptide concentration is about 210 mg/mL.

Embodiment 9: The pharmaceutical composition of any of embodiments 1-8,wherein the N-acetyl arginine is present at a concentration from about25 mM to about 230 mM.

Embodiment 10: The pharmaceutical composition of embodiment 9, whereinthe N-acetyl arginine is present at a concentration from about 140 mM toabout 170 mM

Embodiment 11: The pharmaceutical composition of embodiment 10, whereinthe N-acetyl arginine is present at a concentration of about 140 mM.

Embodiment 12: The pharmaceutical composition of any of embodiments1-11, further comprising a buffer.

Embodiment 13: The pharmaceutical composition of embodiment 13, whereinthe buffer is selected from the group consisting of acetate, glutamate,histidine, and phosphate buffers, or a combination thereof.

Embodiment 14: The pharmaceutical composition of embodiment 13, whereinthe buffer is present at a concentration of about 5 mM to about 30 mM.

Embodiment 15: The pharmaceutical composition of embodiment 14, whereinthe buffer is sodium acetate and is present at a concentration of about10 mM.

Embodiment 16: The pharmaceutical composition of any of embodiments1-15, wherein the pH is about 4.8 to about 6.9.

Embodiment 17: The pharmaceutical composition of embodiment 11, whereinthe pH is about 5.4.

Embodiment 18: The pharmaceutical composition of any of embodiments1-17, further comprising a surfactant.

Embodiment 19: The pharmaceutical composition of embodiment 18, whereinthe surfactant is selected from the group consisting ofpolyoxyethylenesorbitan monooleate (polysorbate 80 or polysorbate 20),polyoxyethylene-polyoxypropylene block copolymer (Poloxamers such asPluronic® F-68 and other Pluronics®), Sorbitan alkyl esters (Spans®)Polyethylene glycol octylphenyl ethers (Triton X-100), Polyethyleneglycol alkyl ethers (Brij), Polypropylene glycol alkyl ethers, Glucosidealkyl ethers, and D-α-tocopherol polyethylene glycol succinate (vitaminE TPGS).

Embodiment 20: The pharmaceutical composition of embodiment 19, whereinthe surfactant is present at a concentration of about 0.0001% (w/v) toabout 1% (w/v).

Embodiment 21: The pharmaceutical composition of embodiment 20, whereinthe surfactant is polyoxyethylenesorbitan monooleate and is present at aconcentration of about 0.01% (w/v).

Embodiment 22: The pharmaceutical composition of any of embodiments1-21, further comprising proline.

Embodiment 23: The pharmaceutical composition of embodiment 22, whereinthe proline is present at a concentration of about 50 mM to about 150mM.

Embodiment 24: The pharmaceutical composition of embodiment 23, whereinthe proline is present at a concentration of about 90 mM to about 120mM.

Embodiment 25: The pharmaceutical composition of embodiment 22 or 23,where the proline is present at a concentration of about a concentrationof about 120 mM.

Embodiment 26: The pharmaceutical composition of any of embodiments1-25, further comprising an arginine salt.

Embodiment 27: The pharmaceutical composition of embodiment 26, whereinthe arginine salt is present at a concentration of about 25 mM to about150 mM.

Embodiment 28: The pharmaceutical composition of embodiment 27, whereinthe arginine salt is present at a concentration of about 50 mM to about100 mM

Embodiment 29: The pharmaceutical composition of embodiment 26, whereinthe arginine salt is arginine-HCl, arginine acetate, or arginineglutamate.

Embodiment 30: The pharmaceutical composition of embodiment 29, whereinthe arginine HCl is present at a concentration of about 50 mM.

Embodiment 31: The pharmaceutical composition of any of embodiments1-30, wherein the PCSK9-binding polypeptide is stable for at least about2 years when stored at about -30° C. or colder.

Embodiment 32: The pharmaceutical composition of embodiment 31, whereinthe PCSK9-binding polypeptide is stable for at least about 5 years.

Embodiment 33: The pharmaceutical composition of any of embodiments1-30, wherein the PCSK9-binding polypeptide is stable for at least about6 months when stored at about 5° C.

Embodiment 34: The pharmaceutical composition of embodiment 33, whereinthe PCSK9-binding polypeptide is stable for at least about 24 months.

Embodiment 35: The pharmaceutical composition of any of embodiments1-30, wherein the PCSK9-binding polypeptide is stable for at least about1 month when stored at about 25° C.

Embodiment 36: The pharmaceutical composition of embodiment 35, whereinthe PCSK9-binding polypeptide is stable for at least about three months.

Embodiment 37: The pharmaceutical composition of embodiment 35, whereinthe PCSK9-binding polypeptide is stable for at least about 6 months.

Embodiment 38: The pharmaceutical composition of any of embodiments1-30, wherein the PCSK9-binding polypeptide is stable for at least about1 month when stored at about 40° C.

Embodiment 39: The pharmaceutical composition of any of embodiments1-38, wherein the composition comprises high molecular weight aggregatesor oligomers of the PCSK9-binding polypeptide at less than about 3% ofthe PCSK9-binding polypeptide concentration.

Embodiment 40: The pharmaceutical composition of embodiment 39, whereinthe high molecular weight aggregates or oligomers of the PCSK9-bindingpolypeptide are present at less than about 2.5% of the PCSK9-bindingpolypeptide concentration.

Embodiment 41: A pharmaceutical composition comprising

-   a. a PCSK9-binding polypeptide that selected from the group    consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, I189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR; and-   b. N-acetyl arginine;-   c. an arginine salt;-   d. a buffer; and-   e. a surfactant-   wherein the pharmaceutical composition has a viscosity of at least    less than about 80 Cp (measured, for example, by a rheometer, such    as TA Instruments (New Castle, DE) AESR-G2 cone and plate    rheometer).

Embodiment 42: The pharmaceutical composition of embodiment 41, whereinthe PCSK9-binding polypeptide is a monoclonal antibody the comprises aheavy chain polypeptide comprising the following complementaritydetermining regions (CDRs):

-   a. heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs:7, 8, and 9, respectively; and-   b. light chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs:4, 5, and 6, respectively.

Embodiment 43: The pharmaceutical composition of embodiment 41 or 42,wherein the pharmaceutical composition has a viscosity of at least lessthan about 50 cP.

Embodiment 44: The pharmaceutical composition of embodiment 41 or 42,wherein the pharmaceutical composition has an osmolality of about 250 toabout 400 mOsm/kg.

Embodiment 45: The pharmaceutical composition of embodiment 44, whereinthe pharmaceutical composition has an osmolality of about 300 mOsm/kg.

Embodiment 46: The pharmaceutical composition of embodiment 45, whereinthe pharmaceutical composition is isotonic to a human blood cell.

Embodiment 47: The pharmaceutical composition of embodiment 41 or 42,wherein the PCSK9-binding polypeptide is present at a concentration ofabout 140 mg/mL to about 260 mg/mL.

Embodiment 48: The pharmaceutical composition of embodiment 47, whereinthe PCSK9-binding polypeptide concentration is about 210 mg/mL.

Embodiment 49: The pharmaceutical composition of any of embodiments41-48, wherein the N-acetyl arginine is present at a concentration fromabout 25 mM to about 230 mM.

Embodiment 50: The pharmaceutical composition of embodiment 49, whereinthe N-acetyl arginine is present at a concentration from about 140 mM toabout 170 mM.

Embodiment 51: The pharmaceutical composition of embodiment 50, whereinthe N-acetyl arginine is present at a concentration of about 140 mM.

Embodiment 52: The pharmaceutical composition of embodiment 41 or 42,wherein the buffer is selected from the group consisting of acetate,glutamate, histidine, and phosphate buffers, or a combination thereof.

Embodiment 53: The pharmaceutical composition of embodiment 52, whereinthe buffer is present at a concentration of about 5 mM to about 30 mM.

Embodiment 54: The pharmaceutical composition of embodiment 53, whereinthe buffer is sodium acetate and is present at a concentration of about10 mM.

Embodiment 55: The pharmaceutical composition of any of embodiments 41or 42, wherein the pH is about 4.8 to about 6.9.

Embodiment 56: The pharmaceutical composition of embodiment 55, whereinthe pH is about 5.4.

Embodiment 57: The pharmaceutical composition of embodiment 41 or 42,wherein the surfactant is selected from the group consisting ofpolyoxyethylenesorbitan monooleate (polysorbate 80 or polysorbate 20),polyoxyethylene-polyoxypropylene block copolymer (Poloxamers such asPluronic® F-68 and other Pluronics®), Sorbitan alkyl esters (Spans®)Polyethylene glycol octylphenyl ethers (Triton X-100), Polyethyleneglycol alkyl ethers (Brij), Polypropylene glycol alkyl ethers, Glucosidealkyl ethers, and D-α-tocopherol polyethylene glycol succinate (vitaminE TPGS).

Embodiment 58: The pharmaceutical composition of embodiment 57, whereinthe surfactant is present at a concentration of about 0.0001% (w/v) toabout 1% (w/v).

Embodiment 59: The pharmaceutical composition of embodiment 57, whereinthe surfactant is polyoxyethylenesorbitan monooleate and is present at aconcentration of about 0.01% (w/v).

Embodiment 60: The pharmaceutical composition of embodiment 41 or 42,further comprising proline.

Embodiment 61: The pharmaceutical composition of embodiment 60, whereinthe proline is present at a concentration of about 50 mM to about 150mM.

Embodiment 62: The pharmaceutical composition of embodiment 61, whereinthe proline is present at a concentration of about 90 mM to about 120mM.

Embodiment 63: The pharmaceutical composition of embodiment 62, wherethe proline is present at a concentration of about a concentration ofabout 120 mM.

Embodiment 64: The pharmaceutical composition of embodiment 41 or 42,wherein the arginine salt is present at a concentration of about 25 mMto about 150 mM.

Embodiment 65: The pharmaceutical composition of embodiment 64, whereinthe arginine salt is present at a concentration of about 50 mM to about100 mM.

Embodiment 66: The pharmaceutical composition of embodiment 64, whereinthe arginine salt is arginine-HCl, arginine acetate, or arginineglutamate.

Embodiment 67: The pharmaceutical composition of embodiment 66, whereinthe arginine HCl is present at a concentration of about 50 mM.

Embodiment 68: The pharmaceutical composition of any of embodiments41-67, wherein the PCSK9-binding polypeptide is stable for at leastabout 2 years when stored at about -30° C. or colder.

Embodiment 69: The pharmaceutical composition of embodiment 68, whereinthe PCSK9-binding polypeptide is stable for at least about 5 years.

Embodiment 70: The pharmaceutical composition of any of embodiments41-67, wherein the PCSK9-binding polypeptide is stable for at leastabout 6 months when stored at about 5° C.

Embodiment 71: The pharmaceutical composition of embodiment 70, whereinthe PCSK9-binding polypeptide is stable for at least about 24 months.

Embodiment 72: The pharmaceutical composition of any of embodiments41-67, wherein the PCSK9-binding polypeptide is stable for at leastabout 1 month when stored at about 25° C.

Embodiment 73: The pharmaceutical composition of embodiment 72, whereinthe PCSK9-binding polypeptide is stable for at least about three months.

Embodiment 74: The pharmaceutical composition of embodiment 73, whereinthe PCSK9-binding polypeptide is stable for at least about 6 months.

Embodiment 75: The pharmaceutical composition of any of embodiments41-67, wherein the PCSK9-binding polypeptide is stable for at leastabout 1 month when stored at about 40° C.

Embodiment 76: The pharmaceutical composition of any of embodiments41-75, wherein the composition comprises high molecular weightaggregates or oligomers of the PCSK9-binding polypeptide at less thanabout 3% of the PCSK9-binding polypeptide concentration.

Embodiment 77: The pharmaceutical composition of embodiment 76, whereinthe high molecular weight aggregates or oligomers of the PCSK9-bindingpolypeptide are present at less than about 2.5% of the PCSK9-bindingpolypeptide concentration.

Embodiment 78: A pharmaceutical composition comprising

-   a. a PCSK9-binding polypeptide that at a concentration of about    195-225 mg/mL selected from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9; iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, I189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR;-   b. N-acetyl arginine present at a concentration of about 140 mM;-   c. arginine HCl present at a concentration of about 50 mM;-   d. polyoxyethylenesorbitan monooleate (polysorbate 80) at a    concentration of from about 0.005% (w/v) to about 0.015% (w/v); and-   e. sodium acetate at a concentration of about 10 mM.

Embodiment 79: The pharmaceutical composition of embodiment 78, whereinthe pharmaceutical composition has a pH of about 5.1 to about 5.7.

Embodiment 80: The pharmaceutical composition of embodiment 78 or 79,wherein the pharmaceutical composition has a viscosity of at least lessthan about 50 Cp (measured, for example, by a rheometer, such as TAInstruments (New Castle, DE) AESR-G2 cone and plate rheometer).

Embodiment 81: A pharmaceutical composition comprising

-   a. a PCSK9-binding polypeptide that at a concentration of about    195-225 mg/mL selected from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, I189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and-   b. wherein the epitope of the monoclonal antibody further overlaps    with a site to which an EGF-A domain of LDLR;-   c. N-acetyl arginine present at a concentration of about 140 mM;-   d. arginine HCl present at a concentration of about 63 mM;-   e. polyoxyethylenesorbitan monooleate (polysorbate 80) at a    concentration of about 0.005% (w/v) to about 0.015%; and-   f. sodium acetate at a concentration of about 10 mM.

Embodiment 82: The pharmaceutical composition of embodiment 81, whereinthe pharmaceutical composition has a pH of about 5.1 to about 5.7.

Embodiment 83: The pharmaceutical composition of embodiment 81 or 82,wherein the pharmaceutical composition has a viscosity of at least lessthan about 80 Cp (measured, for example, by a rheometer, such as TAInstruments (New Castle, DE) AESR-G2 cone and plate rheometer).

Embodiment 84: A pharmaceutical composition comprising

-   a. a PCSK9-binding polypeptide that at a concentration of about    195-225 mg/mL selected from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, I189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR;-   b. N-acetyl arginine present at a concentration of about 155 mM;-   c. arginine HCl present at a concentration of about 70 mM;-   d. polyoxyethylenesorbitan monooleate (polysorbate 80) at a    concentration of about 0.005% (w/v) to about 0.015% (w/v); and-   e. sodium acetate at a concentration of about 10 mM.

Embodiment 85: The pharmaceutical composition of embodiment 84, whereinthe pharmaceutical composition has a pH of about 5.1 to about 5.7.

Embodiment 86: The pharmaceutical composition of embodiment 84 or 85,wherein the pharmaceutical composition has a viscosity of at least lessthan about 45 Cp (measured, for example, by a rheometer, such as TAInstruments (New Castle, DE) AESR-G2 cone and plate rheometer).

Embodiment 87: A pharmaceutical composition comprising

-   a. a PCSK9-binding polypeptide that at a concentration of about    195-225 mg/mL selected from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, I189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR;-   b. N-acetyl arginine present at a concentration of about 170 mM;-   c. arginine HCl present at a concentration of about 63 mM;-   d. polyoxyethylenesorbitan monooleate (polysorbate 80) at a    concentration of about 0.005% (w/v) to about 0.015%; and-   e. sodium acetate at a concentration of about 10 mM.

Embodiment 88: The pharmaceutical composition of embodiment 87, whereinthe pharmaceutical composition has a pH of about 5.1 to about 5.7.

Embodiment 89: The pharmaceutical composition of embodiment 87 or 88,wherein the pharmaceutical composition has a viscosity of at least lessthan about 60 Cp (measured, for example, by a rheometer, such as TAInstruments (New Castle, DE) AESR-G2 cone and plate rheometer).

Embodiment 90: A pharmaceutical composition comprising

-   a. a PCSK9-binding polypeptide that at a concentration of about    195-225 mg/mL selected from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain polypeptide        having an amino acid sequence of SEQ ID NO:1 and a light chain        polypeptide having an amino acid sequence of SEQ ID NO:2        (evolocumab), or an antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. a monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, I189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, and D238, K243, S373,        D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, 1369, S372,        C375, C378, R237, and D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGF-A domain of LDLR;-   b. N-acetyl arginine present at a concentration of about 155 mM;-   c. proline present at a concentration of about 120 mM;-   d. polyoxyethylenesorbitan monooleate (polysorbate 80) at a    concentration of about 0.005% (w/v) to about 0.015% (w/v); and-   e. sodium acetate at a concentration of about 10 mM.

Embodiment 91: The pharmaceutical composition of embodiment 90, whereinthe pharmaceutical composition has a pH of about 5.1 to about 5.7.

Embodiment 92: The pharmaceutical composition of embodiment 90 or 91,wherein the pharmaceutical composition has a viscosity of at least lessthan about 60 Cp (measured, for example, by a rheometer, such as TAInstruments (New Castle, DE) AESR-G2 cone and plate rheometer).

Embodiment 93: The pharmaceutical composition of any of embodiments78-92, wherein the PCSK9-binding polypeptide is stable for at leastabout 2 years when stored at about -30° C. or colder.

Embodiment 94: The pharmaceutical composition of embodiment 93, whereinthe PCSK9-binding polypeptide is stable for at least about 5 years.

Embodiment 95: The pharmaceutical composition of any of embodiments78-92, wherein the PCSK9-binding polypeptide is stable for at leastabout 6 months when stored at about 5° C.

Embodiment 96: The pharmaceutical composition of embodiment 95, whereinthe PCSK9-binding polypeptide is stable for at least about 24 months.

Embodiment 97: The pharmaceutical composition of any of embodiments78-92, wherein the PCSK9-binding polypeptide is stable for at leastabout 1 month when stored at about 25° C.

Embodiment 98: The pharmaceutical composition of embodiment 97, whereinthe PCSK9-binding polypeptide is stable for at least about three months.

Embodiment 99: The pharmaceutical composition of embodiment 98, whereinthe PCSK9-binding polypeptide is stable for at least about 6 months.

Embodiment 100: The pharmaceutical composition of any of embodiments78-92, wherein the PCSK9-binding polypeptide is stable for at leastabout 1 month when stored at about 40° C.

Embodiment 101: The pharmaceutical composition of any of embodiments78-92, wherein the composition comprises high molecular weightaggregates or oligomers of the PCSK9-binding polypeptide at less thanabout 3% of the PCSK9-binding polypeptide concentration.

Embodiment 102: The pharmaceutical composition of embodiment 101,wherein the high molecular weight aggregates or oligomers of thePCSK9-binding polypeptide are present at less than about 2.5% of thePCSK9-binding polypeptide concentration.

Embodiment 103: The pharmaceutical composition of any of embodiments1-102, wherein the pharmaceutical composition is liquid.

Embodiment 104: A method of treating a subject in need thereof,comprising administering the pharmaceutical composition of any ofembodiments 1-103.

Embodiment 105: A kit, comprising the pharmaceutical composition of anyof embodiments 1-103 and a delivery device selected from the groupconsisting of a syringe, an injector pen, a body injector, and anautoinjector.

Embodiment 106: The kit of embodiment 105, further comprisinginstructions for administration of the pharmaceutical composition usingthe delivery device.

Embodiment 107: A method of preparing a PCSK9-binding polypeptide in apharmaceutical composition comprising at least 140 mg/mL ofPCSK9-binding polypeptide, comprising adding to a pharmaceuticalcomposition comprising the PCSK9-binding polypeptide an effective amountof N-acetyl arginine, such that the viscosity of the pharmaceuticalcomposition is reduced when compared to the pharmaceutical compositionlacking the N-acetyl arginine, and wherein the PCSK9-binding polypeptideis selected from the group consisting of:

-   a. a monoclonal antibody comprising a heavy chain polypeptide having    an amino acid sequence of SEQ ID NO:1 and a light chain polypeptide    having an amino acid sequence of SEQ ID NO:2 (evolocumab), or an    antigen-binding fragment thereof;-   b. a monoclonal antibody that competes with evolocumab for binding    to PCSK9;-   c. a monoclonal antibody, comprising:    -   i. a heavy chain polypeptide comprising the following        complementarity determining regions (CDRs): a heavy chain CDR1        that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain CDR2 that        is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain CDR3 that is a        CDR3 in SEQ ID NOs:14 or 16, and    -   ii. a light chain polypeptide comprising the following CDRs: a        light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a light        chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a light chain        CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;        -   d. a monoclonal antibody that binds to at least one of the            following residues of PCSK9, the PCSK9 comprising an amino            acid sequence of SEQ ID NO:3: S153, D188, I189, Q190, S191,            D192, R194, E197, G198, R199, V200, D224, R237, and D238,            K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195,            I196, M201, V202, C223, T228, S235, G236, A239, G244, M247,            1369, S372, C375, C378, R237, and D238; and        -   e. a monoclonal antibody that binds to PCSK9 at an epitope            on PCSK9 that overlaps with an epitope that is bound by an            antibody that comprises:    -   iii. a heavy chain variable region of the amino acid sequence in        SEQ ID NO:1; and    -   iv. a light chain variable region of the amino acid sequence in        SEQ ID NO:2, and    -   v. wherein the epitope of the monoclonal antibody further        overlaps with a site to which an EGF-A domain of LDLR.

Embodiment 108: The method of embodiment 107, wherein the viscosity ofthe pharmaceutical composition is less than 80 Cp (measured, forexample, by a rheometer, such as TA Instruments (New Castle, DE) AESR-G2cone and plate rheometer).

Embodiment 109: The method of embodiment 108, wherein the viscosity ofthe pharmaceutical composition is less than 50 cP.

Embodiment 110: The method of any of embodiments 107-109, wherein thepharmaceutical composition has an osmolality of about 250 to about 400mOsm/kg.

Embodiment 111: The method of embodiment 110, wherein the pharmaceuticalcomposition has an osmolality of about 300 mOsm/kg.

Embodiment 112: The method of embodiment 111, wherein the pharmaceuticalcomposition is isotonic to a human blood cell.

Embodiment 113: The method of embodiment 107, wherein the PCSK9-bindingpolypeptide is present at a concentration of about 180 mg/mL to about260 mg/mL.

Embodiment 114: The method of embodiment 113, wherein the PCSK9-bindingpolypeptide concentration is about 210 mg/mL.

Embodiment 115: The method of any of embodiments 107-114, wherein theN-acetyl arginine is present at a concentration of about 25 mM to about230 mM.

Embodiment 116: The method of embodiment 115, wherein the N-acetylarginine is present at a concentration of about 140 mM to about 170 mM.

Embodiment 117: The method of embodiment 115, wherein the N-acetylarginine is present at a concentration of about 140 mM.

Embodiment 118: The method of any of embodiments 107-117, furthercomprising a buffer.

Embodiment 119: The method of embodiment 118, wherein the buffer isselected from the group consisting of acetate, glutamate, histidine, andphosphate buffers, or a combination thereof.

Embodiment 120: The method of embodiment 119, wherein the buffer ispresent at a concentration of about 5 mM to about 30 mM.

Embodiment 121: The method of embodiment 120, wherein the buffer issodium acetate and is present at a concentration of about 10 mM.

Embodiment 122: The method of any of embodiments 107-121, wherein the pHis about 4.8 to about 6.9. Embodiment 123: The method of embodiment 122,wherein the pH is about 5.4.

Embodiment 124: The method of any of embodiment 107-123, furthercomprising a surfactant.

Embodiment 125: The method of embodiment 124, wherein the surfactant isselected from the group consisting of polyoxyethylenesorbitan monooleate(polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropyleneblock copolymer (Poloxamers such as Pluronic® F-68 and otherPluronics®), Sorbitan alkyl esters (Spans®) Polyethylene glycoloctylphenyl ethers (Triton X-100), Polyethylene glycol alkyl ethers(Brij), Polypropylene glycol alkyl ethers, Glucoside alkyl ethers, andD-α-tocopherol polyethylene glycol succinate (vitamin E TPGS).

Embodiment 126: The method of embodiment 125, wherein the surfactant ispresent at a concentration of about 0.0001% (w/v) to about 1.0% (w/v).

Embodiment 127: The method of embodiment 126, wherein the surfactant ispolyoxyethylenesorbitan monooleate and is present at a concentration ofabout 0.01% (w/v).

Embodiment 128: The method of any of embodiments 107-127, furthercomprising proline.

Embodiment 129: The method of embodiment 128, wherein the proline ispresent at a concentration of about 50 mM to about 150 mM.

Embodiment 130: The method of embodiment 129, wherein the proline ispresent at a concentration of about 90 mM to about 120 mM.

Embodiment 131: The method of embodiment 130, where the proline ispresent at a concentration of about a concentration of about 120 mM.

Embodiment 132: The method of any of embodiments 107-131, furthercomprising an arginine salt.

Embodiment 133: The method of embodiment 132, wherein the arginine saltis present at a concentration of about 25 mM to about 150 mM.

Embodiment 134: The method of embodiment 133, wherein the arginine saltis present at a concentration of about 50 mM to about 100 mM

Embodiment 135: The method of embodiment 133, wherein the arginine saltis arginine-HCl, arginine acetate, or arginine glutamate.

Embodiment 136: The method of embodiment 135, wherein the arginine HClis present at a concentration of about 50 mM.

Embodiment 137: The method of any of embodiments 107-136, wherein thePCSK9-binding polypeptide is stable for at least about 2 years whenstored at about -30° C. or colder.

Embodiment 138: The method of embodiment 137, wherein the PCSK9-bindingpolypeptide is stable for at least about 5 years.

Embodiment 139: The method of any of embodiments 107-136, wherein thePCSK9-binding polypeptide is stable for at least about 6 months whenstored at about 5° C.

Embodiment 140: The method of embodiment 139, wherein the PCSK9-bindingpolypeptide is stable for at least about 24 months.

Embodiment 141: The method of any of embodiments 107-136, wherein thePCSK9-binding polypeptide is stable for at least about 1 month whenstored at about 25° C.

Embodiment 142: The method of embodiment 141, wherein the PCSK9-bindingpolypeptide is stable for at least about three months.

Embodiment 143: The method of embodiment 142, wherein the PCSK9-bindingpolypeptide is stable for at least about 6 months.

Embodiment 144: The method of any of embodiments 107-136, wherein thePCSK9-binding polypeptide is stable for at least about 1 month whenstored at about 40° C.

Embodiment 145: The method of any of embodiments 107-136, wherein thecomposition comprises high molecular weight aggregates or oligomers ofthe PCSK9-binding polypeptide at less than about 3% of the PCSK9-bindingpolypeptide concentration.

Embodiment 146: The method of embodiment 145, wherein the high molecularweight aggregates or oligomers of the PCSK9-binding polypeptide arepresent at less than about 2.5% of the PCSK9-binding polypeptideconcentration.

Embodiment 147: A method of formulating a therapeutic polypeptide,comprising

-   a. a first concentration step, wherein the polypeptide in a first    solution is concentrated;-   b. a first solution exchange step, wherein the concentrated    polypeptide in the first solution is exchanged into a second    solution comprising N-acetyl arginine using diafiltration;-   c. a second concentration step, wherein the polypeptide in the    second solution is concentrated;-   d. a second solution exchange step, wherein the polypeptide in the    concentrated second solution is exchanged into a third solution    comprising N-acetyl arginine using diafiltration; and-   e. a third concentration step, wherein the polypeptide in the third    solution is concentrated;-   wherein the therapeutic polypeptide comprises a PCSK9-binding    polypeptide that blocks binding of PCSK9 to LDLR and is selected    from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain having an        amino acid sequence of SEQ ID NO:1 and a light chain having an        amino acid sequence of SEQ ID NO:2 (evolocumab), or an        antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;            -   i. a monoclonal antibody that binds to at least one of                the following residues of PCSK9, the PCSK9 comprising an                amino acid sequence of SEQ ID NO:3: S153, D188, I189,                Q190, S191, D192, R194, E197, G198, R199, V200, D224,                R237, D238, K243, S373, D374, S376, T377, F379, I154,                T1897, H193, E195, I196, M201, V202, C223, T228, S235,                G236, A239, G244, M247, I369, S372, C375, C378, R237,                D238; ii. a monoclonal antibody that binds to PCSK9 at                an epitope on PCSK9 that overlaps with an epitope that                is bound by an antibody that comprises:        -   3. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   4. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   5. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGFa domain of LDLR.

Embodiment 148: The method of embodiment 147, wherein the PCSK9-bindingpolypeptide that blocks binding of PCSK9 to LDLR is a monoclonalantibody the comprises a heavy chain polypeptide comprising thefollowing complementarity determining regions (CDRs):

-   a. heavy chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs: 7, 8, and 9, respectively; and-   b. light chain CDR1, CDR2, and CDR3 having an amino acid sequence of    SEQ ID NOs: 4, 5, and 6, respectively.

Embodiment 149: The method of embodiment 147, wherein before the thirdconcentration step, the temperature of the solution comprising thepolypeptide is increased from about 25° C. to about 37° C.

Embodiment 150: The method of embodiment 147, wherein the first solutionexchange step is accomplished using at least three diavolumes of thesecond solution.

Embodiment 151: The method of embodiment 147, wherein the secondsolution exchange step is accomplished using at least four diavolumes ofthe third solution.

Embodiment 152: The method of embodiment 147, wherein the initialconcentration of the therapeutic protein is about 11 mg/mL or less.

Embodiment 153: The method of embodiment 147, wherein in the firstconcentration step, the therapeutic polypeptide concentration isincreased from about 3- to about 7-fold.

Embodiment 154: The method of embodiment 153, wherein the increasedconcentration of the polypeptide is from about 35 mg/mL to about 70mg/mL.

Embodiment 155: The method of embodiment 147, wherein in the secondconcentration step, the therapeutic polypeptide concentration isincreased from about 2- to 4-fold from the first concentration step.

Embodiment 156: The method of embodiment 155, wherein the increasedpolypeptide concentration is about 140 mg/mL.

Embodiment 157: The method of embodiment 147, wherein in the thirdconcentration step, the therapeutic polypeptide concentration isincreased from about 1.5- to about 2-fold from the second concentrationstep.

Embodiment 158: The method of embodiment 157, wherein the increasedpolypeptide concentration is about 260 mg/mL.

Embodiment 159: The method of embodiment 147, wherein the therapeuticpolypeptide has a final concentration that is at least about 19-20-foldmore concentrated than the initial concentration of the therapeuticpolypeptide.

Embodiment 160: The method of embodiment 159, wherein the finalconcentration of the therapeutic polypeptide is about 210 mg/mL.

Embodiment 161: The method of embodiment 147, wherein the concentrationsteps comprise fed-batch ultrafiltration.

Embodiment 162: The method of embodiment 147, wherein the secondsolution and the third solution are identical.

Embodiment 163: The method of embodiment 147, wherein the second orthird solution comprising N-acetyl arginine comprises an arginine saltand a buffer.

Embodiment 164: The method of embodiment 163, wherein the N-acetylarginine is present at a concentration of about 25 mM to about 230 mM;the arginine salt is Arg HCl, Arg acetate, or Arg glutamate and ispresent at a concentration of about 25 mM to about 150 mM; and thebuffer is a sodium acetate buffer at a concentration of about 5 mM toabout 30 mM.

Embodiment 165: The method of embodiment 164, wherein the N-acetylarginine is present at a concentration of about 140 to about 170 mM; theArg HCl, Arg acetate, or Arg glutamate is present at a concentration ofabout 63 to about 70 mM and the sodium acetate buffer is present at aconcentration of about 10 mM.

Embodiment 166: The method of embodiment 164, wherein the N-acetylarginine is present at a concentration of about 140 mM, the Arg HCl, Argacetate, or Arg glutamate is present at a concentration of about 63 mM,the sodium acetate buffer is present at a concentration of about 10 mM.

Embodiment 167: The method of embodiment 164, wherein the N-acetylarginine is present at a concentration of about 155 mM, the Arg HCl, Argacetate, or Arg glutamate is present at a concentration of about 70 mM,the sodium acetate buffer is present at a concentration of about 10 mM.

Embodiment 168: The method of embodiment 164, wherein the N-acetylarginine is present at a concentration of about 170 mM, the Arg HCl, Argacetate, or Arg glutamate is present at a concentration of about 63 mM,the sodium acetate buffer is present at a concentration of about 10 mM.

Embodiment 169: The method of embodiment 165 or 166, further comprisingproline, wherein the proline is present at a concentration of about 50mM to about 150 mM.

Embodiment 170: The method of any of embodiments 163-169, wherein thesecond or third solution has a pH from about 4.8 to about 6.9.

Embodiment 171: The method of embodiment 170, wherein the pH is about5.4.

Embodiment 172: The method of embodiment 147, wherein in the first andsecond solution exchange steps, a diafiltration membrane is used havingat least one characteristic selected from the group consisting of:

-   a. mesh openings that are greater than about 350 µm but less than or    equal to about 500 µm;-   b. an open area that is greater than about 32% but less than or    equal to about 36% of the membrane area;-   c. a mesh count of less than about 16.2 n/cm but greater than or    equal to about 12.2 n/cm;-   d. a wire diameter that is greater than about 270 µm but less than    or equal to about 340 µm;-   e. a basis weight that is greater than about 160 g/m² but less than    or equal to 180 g/m²;-   f. a thickness greater than about 515 µm but less than or equal to    about 610 µm;-   g. a membrane load of greater than about 1138.1 g/m² but less than    or equal to about 1919.3 g/m²; and-   h. a maximum feed pressure of about 60 psi.

Embodiment 173: The method of any of embodiments 147-172, whereinsurfactant is added to the third solution after being concentrated.

Embodiment 174: The method of embodiment 173, wherein the surfactant isselected from the group consisting of polyoxyethylenesorbitan monooleate(polysorbate 80 or polysorbate 20), polyoxyethylene-polyoxypropyleneblock copolymer (Poloxamers such as Pluronic® F-68 and otherPluronics®), Sorbitan alkyl esters (Spans®) Polyethylene glycoloctylphenyl ethers (Triton X-100), Polyethylene glycol alkyl ethers(Brij), Polypropylene glycol alkyl ethers, Glucoside alkyl ethers, andD-α-tocopherol polyethylene glycol succinate (vitamin E TPGS) and ispresent at a concentration of about 0.0001% to about 1%.

Embodiment 175: The method of embodiment 175, wherein the surfactant ispolyoxyethylenesorbitan monooleate (polysorbate 80) and is present at aconcentration of about 0.01% (w/v).

Embodiment 176: A method of formulating a therapeutic polypeptide,comprising

-   a. a first concentration step, wherein the polypeptide in a first    solution is concentrated using fed-batch ultrafiltration;-   b. a first solution exchange step, wherein the concentrated    polypeptide in the first solution is exchanged into a second    solution comprising N-acetyl arginine, arginine salt, and a buffer,    using diafiltration and three diavolumes of the second solution;-   c. a second concentration step, wherein the polypeptide in the    second solution is concentrated using fed-batch ultrafiltration;-   d. a second solution exchange step, wherein the polypeptide in the    concentrated second solution is exchanged into third solution    comprising N-acetyl arginine, arginine salt, and a buffer using    diafiltration and four diavolumes of the third solution;-   e. the temperature of the solution comprising the polypeptide is    increased from about 25° C. to about 37° C. after the second    solution exchange step; and-   f. a third concentration step, wherein the polypeptide is further    concentrated using fed-batch ultrafiltration concentration;-   wherein in the first and second solution exchange steps, a    diafiltration membrane is used having at least one characteristic    selected from the group consisting of:-   g. mesh openings that are greater than about 350 µm but less than or    equal to about 500 µm;-   h. an open area that is greater than about 32% but less than or    equal to about 36% of the membrane area;-   i. a mesh count of less than about 16.2 n/cm but greater than or    equal to about 12.2 n/cm;-   j. a wire diameter that is greater than about 270 µm but less than    or equal to about 340 µm;-   k. a basis weight that is greater than about 160 g/m² but less than    or equal to 180 g/m²;-   l. a thickness greater than about 515 µm but less than or equal to    about 610 µm;-   m. a membrane load of greater than about 1138.1 g/m² but less than    or equal to about 1919.3 g/m²; and-   n. a maximum feed pressure of about 60 psi; and-   wherein the therapeutic polypeptide comprises a PCSK9-binding    polypeptide that blocks binding of PCSK9 to LDLR and is selected    from the group consisting of:    -   i. a monoclonal antibody comprising a heavy chain having an        amino acid sequence of SEQ ID NO:1 and a light chain having an        amino acid sequence of SEQ ID NO:2 (evolocumab), or an        antigen-binding fragment thereof;    -   ii. a monoclonal antibody that competes with evolocumab for        binding to PCSK9;    -   iii. a monoclonal antibody, comprising:        -   1. a heavy chain polypeptide comprising the following            complementarity determining regions (CDRs): a heavy chain            CDR1 that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain            CDR2 that is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain            CDR3 that is a CDR3 in SEQ ID NOs:14 or 16, and        -   2. a light chain polypeptide comprising the following CDRs:            a light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a            light chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a            light chain CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;    -   iv. monoclonal antibody that binds to at least one of the        following residues of PCSK9, the PCSK9 comprising an amino acid        sequence of SEQ ID NO:3: S153, D188, I189, Q190, S191, D192,        R194, E197, G198, R199, V200, D224, R237, D238, K243, S373,        D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201,        V202, C223, T228, S235, G236, A239, G244, M247, I369, S372,        C375, C378, R237, D238;    -   v. a monoclonal antibody that binds to PCSK9 at an epitope on        PCSK9 that overlaps with an epitope that is bound by an antibody        that comprises:        -   1. a heavy chain variable region of the amino acid sequence            in SEQ ID NO:1; and        -   2. a light chain variable region of the amino acid sequence            in SEQ ID NO:2, and        -   3. wherein the epitope of the monoclonal antibody further            overlaps with a site to which an EGFa domain of LDLR.

Embodiment 177: The method of embodiment 176, wherein before the thirdconcentration step, the temperature of the solution comprising thepolypeptide is increased from about 25° C. to about 37° C.

Embodiment 178: The method of embodiment 176, wherein the initialconcentration of the therapeutic protein is 11 mg/mL or less.

Embodiment 179: The method of embodiment 176, wherein in the firstconcentration step, the therapeutic polypeptide concentration isincreased from about 3- to about 7-fold.

Embodiment 180: The method of embodiment 179, wherein the increasedconcentration of the polypeptide is from about 35 mg/mL to about 70mg/mL.

Embodiment 181: The method of embodiment 176, wherein in the secondconcentration step, the therapeutic polypeptide concentration isincreased about 2- to 4-fold from the first concentration step.

Embodiment 182: The method of embodiment 181, wherein the increasedpolypeptide concentration is about 140 mg/mL.

Embodiment 183: The method of embodiment 176, wherein in the thirdconcentration step, the therapeutic polypeptide concentration isincreased from about 1.5- to about 2-fold from the second concentrationstep.

Embodiment 184: The method of embodiment 183, wherein the increasedpolypeptide concentration is about 260 mg/mL.

Embodiment 185: The method of embodiment 176, wherein the therapeuticpolypeptide has a final concentration that is at least about 19-20-foldmore concentrated than the initial concentration of the therapeuticpolypeptide.

Embodiment 186: The method of embodiment 185, wherein the finalconcentration of the therapeutic polypeptide is about 210 mg/mL.

Embodiment 187: The method of embodiment 176, wherein the N-acetylarginine is present at a concentration of about 25 mM to about 230 mM;the arginine salt is Arg HCl, Arg acetate, or Arg glutamate and ispresent at a concentration of about 25 mM to about 150 mM, and thebuffer is a sodium acetate buffer at a concentration of about 5 mM toabout 30 mM.

Embodiment 188: The method of embodiment 188, wherein the N-acetylarginine is present at a concentration of about 140 to about 170 mM; theArg HCl, Arg acetate, or Arg glutamate is present at a concentration ofabout 63 to about 70 mM and the sodium acetate buffer is present at aconcentration of about 10 mM.

Embodiment 189: The method of embodiment 188, wherein the N-acetylarginine is present at a concentration of about 140 mM, the Arg HCl, Argacetate, or Arg glutamate is present at a concentration of about 63 mM,the sodium acetate buffer is present at a concentration of about 10 mM.

Embodiment 190: The method of embodiment 188, wherein the N-acetylarginine is present at a concentration of about 155 mM, the Arg HCl, Argacetate, or Arg glutamate is present at a concentration of about 70 mM,the sodium acetate buffer is present at a concentration of about 10 mM.

Embodiment 191: The method of embodiment 188, wherein the N-acetylarginine is present at a concentration of about 170 mM, the Arg HCl, Argacetate, or Arg glutamate is present at a concentration of about 63 mM,the sodium acetate buffer is present at a concentration of about 10 mM.

Embodiment 192: The method of embodiment 176, further comprisingproline, wherein the proline is present at a concentration of about 50mM to about 150 mM.

Embodiment 193: The method of any of embodiments 176-192, wherein thesecond or third solution has a pH from about 4.8 to about 6.9.

Embodiment 194: The method of embodiment 193, wherein the pH is about5.4.

Embodiment 195: The method of any of embodiments 176-194, whereinsurfactant is added to the third solution after being concentrated.

Embodiment 196: The method of embodiment 195, wherein the surfactant iswherein the surfactant is selected from the group consisting ofpolyoxyethylenesorbitan monooleate (polysorbate 80 or polysorbate 20),polyoxyethylene-polyoxypropylene block copolymer (Poloxamers such asPluronic® F-68 and other Pluronics®), Sorbitan alkyl esters (Spans®)Polyethylene glycol octylphenyl ethers (Triton X-100), Polyethyleneglycol alkyl ethers (Brij), Polypropylene glycol alkyl ethers, Glucosidealkyl ethers, and D-α-tocopherol polyethylene glycol succinate (vitaminE TPGS) and is present at a concentration of about 0.0001% to about 1%.

Embodiment 197: The method of embodiment 196, wherein the surfactant ispolyoxyethylenesorbitan monooleate (polysorbate 80) and is present at aconcentration of about 0.01% (w/v).

Embodiment 198: A method of formulating a therapeutic polypeptide,comprising

-   a. a first concentration step, wherein the polypeptide in a first    solution is concentrated using fed-batch ultrafiltration;-   b. a first solution exchange step, wherein the concentrated    polypeptide in the first solution is exchanged into a second using    diafiltration and three diavolumes of the second solution;-   c. a second concentration step, wherein the polypeptide in the    second solution is concentrated using fed-batch ultrafiltration;-   d. a second solution exchange step, wherein the polypeptide in the    concentrated second solution is exchanged into third solution using    diafiltration and four diavolumes of the third solution;-   e. the temperature of the solution comprising the polypeptide is    increased from about 25° C. to about 37° C. after the second    solution exchange step; and-   f. a third concentration step, wherein the polypeptide is further    concentrated using fed-batch ultrafiltration concentration;-   g. alternatively, a step adding polyoxyethylenesorbitan monooleate    at a concentration of about 0.01% (w/v) to the resulting solution of    the third concentration step,-   wherein the second and third solutions comprise a solution selected    from the group consisting of a solution comprising about 140 mM    N-acetyl arginine, about 50 mM Arg HCl, and about 10 mM sodium    acetate, the solution having a pH of about 5.2; a solution    comprising about 155 mM N-acetyl arginine, about 70 mM Arg HCl, and    about 10 mM sodium acetate, the solution having a pH of about 5.4;    and a solution comprising about 170 mM N-acetyl arginine, about 10    mM sodium acetate, the solution having a pH of about 5.6;-   wherein in the first and second solution exchange steps, a    diafiltration membrane is used having at least one characteristic    selected from the group consisting of:-   h. mesh openings that are greater than about 350 µm but less than or    equal to about 500 µm;-   i. an open area that is greater than about 32% but less than or    equal to about 36% of the membrane area;-   j. a mesh count of less than about 16.2 n/cm but greater than or    equal to about 12.2 n/cm;-   k. a wire diameter that is greater than about 270 µm but less than    or equal to about 340 µm;-   l. a basis weight that is greater than about 160 g/m² but less than    or equal to 180 g/m²;-   m. a thickness greater than about 515 µm but less than or equal to    about 610 µm;-   n. a membrane load of greater than about 1138.1 g/m² but less than    or equal to about 1919.3 g/m²; and-   o. a maximum feed pressure of about 60 psi; and

wherein the therapeutic polypeptide comprises a PCSK9-bindingpolypeptide that blocks binding of PCSK9 to LDLR and is selected fromthe group consisting of:

-   i. a monoclonal antibody comprising a heavy chain having an amino    acid sequence of SEQ ID NO:1 and a light chain having an amino acid    sequence of SEQ ID NO:2 (evolocumab), or an antigen-binding fragment    thereof;-   ii. a monoclonal antibody that competes with evolocumab for binding    to PCSK9;-   iii. a monoclonal antibody, comprising:    -   1. a heavy chain polypeptide comprising the following        complementarity determining regions (CDRs): a heavy chain CDR1        that is a CDR1 in SEQ ID NOs:14 or 16; a heavy chain CDR2 that        is a CDR2 in SEQ ID NOs:14 or 16; a heavy chain CDR3 that is a        CDR3 in SEQ ID NOs:14 or 16, and    -   2. a light chain polypeptide comprising the following CDRs: a        light chain CDR1 that is a CDR1 in SEQ ID NOs:15 or 17; a light        chain CDR2 that a CDR2 in SEQ ID NOs:15 or 17; and a light chain        CDR3 that is a CDR3 in SEQ ID NOs:15 or 17;-   iv. a monoclonal antibody that binds to at least one of the    following residues of PCSK9, the PCSK9 comprising an amino acid    sequence of SEQ ID NO:3: S153, D188, I189, Q190, S191, D192, R194,    E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376,    T377, F379, I154, T1897, H193, E195, I196, M201, V202, C223, T228,    S235, G236, A239, G244, M247, I369, S372, C375, C378, R237, D238;-   v. a monoclonal antibody that binds to PCSK9 at an epitope on PCSK9    that overlaps with an epitope that is bound by an antibody that    comprises:    -   1. a heavy chain variable region of the amino acid sequence in        SEQ ID NO:1; and    -   2. a light chain variable region of the amino acid sequence in        SEQ ID NO:2, and

wherein the epitope of the monoclonal antibody further overlaps with asite to which an EGFa domain of LDLR.

Embodiment 199: The method of embodiment 104, wherein the subject has adisease or disorder selected from the group consisting of

-   a cholesterol related disease or disorder selected from the group    consisting of familial hypercholesterolemia (including including    heterozygous hypercholesterolemia, homozygous hypercholesterolemia,    familial defective apop lipoprotein B-100; polygenic    hypercholesterolemia), non-familial hypercholesterolemia,    hyperlipidemia, heart disease, metabolic syndrome, diabetes,    coronary heart disease, stroke, cardiovascular diseases, Alzheimer’s    disease and dyslipidemias (including primary and secondary    dyslipidemias, such as metabolic syndrome, diabetes mellitus,    familial combined hyperlipidemia, familial hypertriglyceridemia;    remnant removal disease, hepatic lipase deficiency; dyslipidemia    secondary to dietary indiscretion, hypothyroidism, drugs including    estrogen and progestin therapy, beta-blockers, and thiazide    diuretics; nephrotic syndrome, chronic renal failure, Cushing’s    syndrome, primary biliary cirrhosis, glycogen storage diseases,    hepatoma, cholestasis, acromegaly, insulinoma, isolated growth    hormone deficiency, or alcohol-induced hypertriglyceridemia.-   atherosclerotic disease selected from the group consisting of    cardiovascular death, non-cardiovascular or all-cause death,    coronary heart disease, coronary artery disease, peripheral arterial    disease, stroke (ischaemic and hemorrhagic), angina pectoris,    cerebrovascular disease and acute coronary syndrome, myocardial    infarction and unstable angina; and-   a disease or disorder that can be addressed using statins.

Embodiment 200: The method of embodiment 104, wherein the treating thesubject comprises reducing the risk of a condition selected from thegroup consisting of fatal and nonfatal heart attack, fatal and non-fatalstroke, heart surgery, hospitalization for heart failure, chest pain ina subject having heart disease, and/or cardiovascular events because ofestablished heart disease, cardiovascular condition due to elevated CRPor hsCRP, and a recurrent cardiovascular event.

Embodiment 201: The pharmaceutical composition of any of embodiments1-103 for use as a medicament.

Embodiment 202: The pharmaceutical composition of embodiment 201,wherein the medicament is for use in the treatment of a disease ordisorder selected from the group consisting of

-   a cholesterol related disease or disorder selected from the group    consisting of familial hypercholesterolemia (including including    heterozygous hypercholesterolemia, homozygous hypercholesterolemia,    familial defective apop lipoprotein B-100; polygenic    hypercholesterolemia), non-familial hypercholesterolemia,    hyperlipidemia, heart disease, metabolic syndrome, diabetes,    coronary heart disease, stroke, cardiovascular diseases, Alzheimer’s    disease and dyslipidemias (including primary and secondary    dyslipidemias, such as metabolic syndrome, diabetes mellitus,    familial combined hyperlipidemia, familial hypertriglyceridemia;    remnant removal disease, hepatic lipase deficiency; dyslipidemia    secondary to dietary indiscretion, hypothyroidism, drugs including    estrogen and progestin therapy, beta-blockers, and thiazide    diuretics; nephrotic syndrome, chronic renal failure, Cushing’s    syndrome, primary biliary cirrhosis, glycogen storage diseases,    hepatoma, cholestasis, acromegaly, insulinoma, isolated growth    hormone deficiency, or alcohol-induced hypertriglyceridemia.-   atherosclerotic disease selected from the group consisting of    cardiovascular death, non-cardiovascular or all-cause death,    coronary heart disease, coronary artery disease, peripheral arterial    disease, stroke (ischaemic and hemorrhagic), angina pectoris,    cerebrovascular disease and acute coronary syndrome, myocardial    infarction and unstable angina; and-   a disease or disorder that can be addressed using statins.

Embodiment 203: The method of embodiment 202, wherein the medicament isfor to reduce the risk of a condition selected from the group consistingof fatal and nonfatal heart attack, fatal and non-fatal stroke, heartsurgery, hospitalization for heart failure, chest pain in a subjecthaving heart disease, and/or cardiovascular events because ofestablished heart disease, cardiovascular condition due to elevated CRPor hsCRP, and a recurrent cardiovascular event.

ABBREVIATIONS Abbreviation Definition Arg HCl arginine HCl ASC Ambientstorage conditions CDR Complementary determining region CEX Cationexchange DF Diafiltration DIW Deionized water DOE Design of ExperimentDS Drug substance DV Diavolume EGF-A Epidermal growth factor-like repeatA EQ Equilibrium FR Framework region HC Heavy chain (antibody) HCVRHeavy chain variable region (antibody) HDLC High-density lipoproteincholesterol HIAC Subvisible particle detection by light obscuration HMWHigh molecular weight HPLC High-pressure liquid chromatography HPLC-UVHigh pressure liquid chromatography ultraviolet LC Light chain(antibody) LCVR Light chain variable region (antibody) LDL Low-densitylipoprotein LDLR Low-density lipoprotein receptors LMH Liters/m²/hr LMWLow molecular weight mAb Monoclonal antibody MFI Micro-flow imagingNaOAC Sodium acetate NAR N-acetyl arginine NTU Nephelometric TurbidityUnit OC Over-concentrated, Over-concentration PEG Polyethylene glycolPFS Pre-filled syringe PVDF Polyvinylidene fluoride PW Purified waterrCE-SDS Reduced capillary electrophoresis - sodium dodecyl sulfate RTRoom temperature SEC Size-exclusion chromatography SE-HPLCSize-exclusion high pressure liquid chromatography TFF Tangential flowfiltration TMP Transmembrane pressure UF Ultrafiltration UFDFUltrafiltration/diafiltration UV Ultraviolet VF Viral filtration VLDL-CVery-low-density lipoprotein cholesterol WFI Water for injection

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1-13. (canceled)
 14. A method of preparing a PCSK9-binding polypeptidein a pharmaceutical composition comprising at least 140 mg/mL ofPCSK9-binding polypeptide, comprising adding to a pharmaceuticalcomposition comprising the PCSK9-binding polypeptide an effective amountof N-acetyl arginine, such that the viscosity of the pharmaceuticalcomposition is reduced when compared to the pharmaceutical compositionlacking the N-acetyl arginine, and wherein the PCSK9-binding polypeptideis selected from the group consisting of: a. a monoclonal antibodycomprising a heavy chain polypeptide having an amino acid sequence ofSEQ ID NO: 1 and a light chain polypeptide having an amino acid sequenceof SEQ ID NO:2 (evolocumab), or an antigen-binding fragment thereof; b.a monoclonal antibody that competes with evolocumab for binding toPCSK9; c. a monoclonal antibody, comprising: i. a heavy chainpolypeptide comprising the following complementarity determining regions(CDRs): a heavy chain CDR1 that is a CDR1 in SEQ ID NOs: 14 or 16; aheavy chain CDR2 that is a CDR2 in SEQ ID NOs: 14 or 16; a heavy chainCDR3 that is a CDR3 in SEQ ID NOs: 14 or 16, and ii. a light chainpolypeptide comprising the following CDRs: a light chain CDR1 that is aCDR1 in SEQ ID NOs: 15 or 17; a light chain CDR2 that a CDR2 in SEQ IDNOs: 15 or 17; and a light chain CDR3 that is a CDR3 in SEQ ID NOs: 15or 17; d. a monoclonal antibody that binds to at least one of thefollowing residues of PCSK9, the PCSK9 comprising an amino acid sequenceof SEQ ID NO:3: S153, D188, I189, Q190, S191, D192, R194, E197, G198,R199, V200, D224, R237, and D238, K243, S373, D374, S376, T377, F379,I154, T1897, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239,G244, M247, I369, S372, C375, C378, R237, and D238; and e. a monoclonalantibody that binds to PCSK9 at an epitope on PCSK9 that overlaps withan epitope that is bound by an antibody that comprises: i. a heavy chainvariable region of the amino acid sequence in SEQ ID NO:1; and ii. alight chain variable region of the amino acid sequence in SEQ ID NO:2,and iii. wherein the epitope of the monoclonal antibody further overlapswith a site to which an EGF-A domain of LDLR.
 15. A method offormulating a therapeutic polypeptide, comprising a. a firstconcentration step, wherein the polypeptide in a first solution isconcentrated; b. a first solution exchange step, wherein theconcentrated polypeptide in the first solution is exchanged into asecond solution comprising N-acetyl arginine using diafiltration; c. asecond concentration step, wherein the polypeptide in the secondsolution is concentrated; d. a second solution exchange step, whereinthe polypeptide in the concentrated second solution is exchanged into athird solution comprising N-acetyl arginine using diafiltration; and e.a third concentration step, wherein the polypeptide in the thirdsolution is concentrated; wherein the therapeutic polypeptide comprisesa PCSK9-binding polypeptide that blocks binding of PCSK9 to LDLR and isselected from the group consisting of: i. a monoclonal antibodycomprising a heavy chain having an amino acid sequence of SEQ ID NO: 1and a light chain having an amino acid sequence of SEQ ID NO:2(evolocumab), or an antigen-binding fragment thereof; ii. a monoclonalantibody that competes with evolocumab for binding to PCSK9; iii. amonoclonal antibody, comprising:
 1. a heavy chain polypeptide comprisingthe following complementarity determining regions (CDRs): a heavy chainCDR1 that is a CDR1 in SEQ ID NOs: 14 or 16; a heavy chain CDR2 that isa CDR2 in SEQ ID NOs: 14 or 16; a heavy chain CDR3 that is a CDR3 in SEQID NOs: 14 or 16, and
 2. a light chain polypeptide comprising thefollowing CDRs: a light chain CDR1 that is a CDR1 in SEQ ID NOs: 15 or17; a light chain CDR2 that a CDR2 in SEQ ID NOs: 15 or 17; and a lightchain CDR3 that is a CDR3 in SEQ ID NOs: 15 or 17; iv. a monoclonalantibody that binds to at least one of the following residues of PCSK9,the PCSK9 comprising an amino acid sequence of SEQ ID NO:3: S153, D188,I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237, D238,K243, S373, D374, S376, T377, F379, I154, T1897, H193, E195, I196, M201,V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375, C378,R237, D238; v. a monoclonal antibody that binds to PCSK9 at an epitopeon PCSK9 that overlaps with an epitope that is bound by an antibody thatcomprises:
 1. a heavy chain variable region of the amino acid sequencein SEQ ID NO:1; and
 2. a light chain variable region of the amino acidsequence in SEQ ID NO:2, and
 3. wherein the epitope of the monoclonalantibody further overlaps with a site to which an EGFa domain of LDLR.16. The method of claim 0, wherein the PCSK9-binding polypeptide thatblocks binding of PCSK9 to LDLR is a monoclonal antibody the comprises aheavy chain polypeptide comprising the following complementaritydetermining regions (CDRs): a. heavy chain CDR1, CDR2, and CDR3 havingan amino acid sequence of SEQ ID NOs: 7, 8, and 9, respectively; and b.light chain CDR1, CDR2, and CDR3 having an amino acid sequence of SEQ IDNOs: 4, 5, and 6, respectively.
 17. The method of claim 0, whereinbefore the third concentration step, the temperature of the solutioncomprising the polypeptide is increased from about 25° C. to about 37°C.
 18. The method of claim 0, wherein in the first concentration step,the therapeutic polypeptide concentration is increased from about 3- toabout 7-fold.
 19. The method of claim 0, wherein in the secondconcentration step, the therapeutic polypeptide concentration isincreased from about 2- to 4-fold from the first concentration step. 20.The method of claim 0, wherein in the third concentration step, thetherapeutic polypeptide concentration is increased from about 1.5- toabout 2-fold from the second concentration step.
 21. The method of claim0, wherein the therapeutic polypeptide has a final concentration that isat least about 19-20-fold more concentrated than the initialconcentration of the therapeutic polypeptide.
 22. The method of claim 0,wherein the concentration steps comprise fed-batch ultrafiltration. 23.The method of claim 15, wherein the second or third solution has a pHfrom about 4.8 to about 6.9.
 24. The method of claim 0, wherein in thefirst and second solution exchange steps, a diafiltration membrane isused having at least one characteristic selected from the groupconsisting of: a. mesh openings that are greater than about 350 µm butless than or equal to about 500 µm; b. an open area that is greater thanabout 32% but less than or equal to about 36% of the membrane area; c. amesh count of less than about 16.2 n/cm but greater than or equal toabout 12.2 n/cm; d. a wire diameter that is greater than about 270 µmbut less than or equal to about 340 µm; e. a basis weight that isgreater than about 160 g/m² but less than or equal to 180 g/m2; f. athickness greater than about 515 µm but less than or equal to about 610µm; g. a membrane load of greater than about 1138.1 g/m² but less thanor equal to about 1919.3 g/m²; and h. a maximum feed pressure of about60 psi.
 25. The method of claim 15, wherein surfactant is added to thethird solution after being concentrated.